转TsVP提高玉米低磷耐受性的研究及不同玉米基因型低磷响应microRNA的差异分析
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摘要
磷是植物生长发育不可缺少的大量营养元素之一。然而土壤有效磷浓度很低,远不能满足植物获取充足磷营养的需求。玉米是重要的粮食、饲料和能源作物。土壤有效磷不足是限制玉米产量的重要因素。向土壤中大量施加磷肥能减少有效磷不足造成的产量损失,但是磷肥投入不仅大大增加生产成本,而且严重污染环境。因此深入研究玉米耐低磷机制,发展耐低磷玉米品种,增加玉米在低磷条件下的产量,地农业、经济和环境都有重要的意义。
转TsVP提高玉米低磷耐受性的研究
通过植物基因工程的手段将有价值的基因转入玉米是发展耐低磷玉米品种的重要途径。许多研究表明超表达氢离子焦磷酸酶(H+-PPase)基因能够在多种植物中促进植物根系生长发育并能提高植物的抗逆性。在高等植物中,根系是植物吸收矿质营养的主要器官,尤其磷素在土壤中的移动性很低,所以根系的生长发育和形态结构对捕获吸收土壤磷素和提高植物耐低磷能力尤为重要。因此为了探索H+-PPase能否提高玉米的耐低磷能力,本工作中我们选用玉米自交系DH4866以及其转盐芥H+-PPase基因(TsVP)玉米为实验材料,比较两者的耐低磷能力以及在低磷条件下的产量,以期得到耐低磷能力提高的转基因玉米材料,为培养耐低磷玉米品种提供育种材料。
将玉米在足磷(sufficient phosphate, SP,1,000μM KH2PO4)营养液中培养20天,然后一半植株在足磷营养液中培养,另一半株在低磷(low phosphate, LP,5μM KH2PO4)营养液中培养,共同培养25天。低磷条件下,转基因玉米生长发育受抑制的程度低于非转基因植株,其地上部分生物量、根系生量和植株生物量都显著高于非转基因植株(P<0.05),说明转TsVP玉米有较高的耐低磷能力。无论在正常生长条件下还是低磷胁迫条件下,转基因玉米的根系都比非转基因植株发达,其根系生物量干重、根冠比、侧根数目、侧根长度、总根长和根系吸收面积都显著高于非转基因植株(P<005),说明在玉米中超表达TsVP能够显著促进玉米根系生长发育。转基因玉米的茎叶和根部可溶性总糖含量和可溶性总糖含量根冠比与非转基因植株相比都没有显著差异(P>0.05),说明碳水化合物的含量和分配不是造成转基因玉米和非转基因玉米根系差异的原因。无论在足磷条件下还是在低磷条件下,转基因玉米植株生长素总量非转基因植株相比无差别,但是根系生长素含量明显高于非转基因植株,而茎叶生长素含量明显低于非转基因植株,说明在玉米中超表达TsFP能够促进生长素向根系分配。转基因玉米中几个生长素运输相关基因(ZmAU.X1、ZmPIN1a和ZmPIN1b)的表达水平显著高于非转基因植株(P<005),说明过表达TsVP可能影响了玉米植株体内生长素运输过程。对转基因玉米和非转基因玉米都外源施加IAA或NPA后,它们在根系生物量和根系形态参数方面的显著性差异消失。这些结果都说明转基因玉米和非转基因玉米之间在根系方面的差异涉及生长素的参与,说明在玉米中超表达TsVP可能是通过促进生长素向根部运输从而促进转基因玉米根系发达。
不论在足磷条件下还是低磷条件下,转基因玉米的最大吸收速率Imax值都显著高于非转基因植株(P<005),但是(min和Km值与非转基因植株相比无显著差异(P>0.05),说明玉米磷转运体对磷的亲和力不是导致转基因玉米与非转基因玉米磷吸收速率差异的原因,因此推测转基因玉米有较高的吸收速率与其有较发达根系系统有关。转基因玉米茎叶和磷含量都显著高于非转基因植株(P<0.05),这与其有较高的磷吸收速率相一致。较高的磷浓度对低磷胁迫下的植株的生长发育极有利,这可能是低磷条件下转基因玉米生长发育受低磷抑制的程度较低的原因。
转基因玉米培养液的pH值显著低于非转基因植株(P<005),表明其根际酸化能力高于非转基因植株。但是其根系有机酸分泌速率与和非转基因玉米相比无显著差异(P>0.05),说明有机酸分泌速率并不是转基因玉米有较强根际酸化能力的原因。但昌无论在是磷条件还是在低磷条件下,转基因玉米的P*ATPase酶活性都显著高于非转基因植株(P<005),说明在米中超表达TsVP能够通过提高P*ATPase(?)活性从面提高玉米根际酸化能力。
无论在足磷条件下还是低磷条件下转基因玉米和非转基因植株相应部位的组织内酸性磷酸酶活性外泌酸性磷酸酶的活性均没有显著差异(P>005)。说明超表达TsVP并没有改变玉米的酸性磷酸酶活性,其并不是转基因玉米耐低磷能力提高的原因。
以上结果表明在玉米中超表达盐芥H-PPase基因TsVP可能通过促进生长素向根中运输来促进玉米根系生长发育而且能够能过诱导细胞膜H-ATPase来提高玉米根际酸化能力。,这些特征都与植物适应低磷胁迫相关,根系发达以及根际酸化能力增强都有利于植物在低磷条件下的生长。因此推测较发达的根系系统和较高的根际酸化能力是转TsVP玉米耐低磷能力提高的原因。
在低磷土壤中,转基因玉米在不同时时期的生长发育受低磷抑制的程度均低于非转基因玉米,其茎叶生物理、根系生物量、植株生物量和生物量的根冠比在各个生长阶段都显著高于非转基因玉米(P<0.05),说明在玉米中超表达TsVP在各个生长阶段都能促进玉米根系的生长发育和提高玉米的耐低磷能力。在低磷土壤中生长的转基因玉米的磷浓度和磷含量显著高于非转基因玉米(P<0.05),这与其有较发达的根系和较高的根际酸化能力相一致,而较高的磷浓度又有利于植物在低磷条件下生长发育,这可能是低磷土壤中转基因玉米生长发育受低磷胁迫抑制的程度低于非转基因玉米的原因。
在低磷土壤中生长的玉米的净光合值和气孔导度明显显低于在足磷土壤中生长的玉米,而胞间二氧化碳浓度明显高于在足磷土壤中生长的玉米,说明低磷胁迫造成的植物光合速率下降主要是由非气孔因素引起的。在低磷土壤中,转基因玉米的净光合值和气孔导度暄著高于非转基因植株(P<0.05),而胞间二氧化碳浓度显著低于非转基因植株(P<0.05),说明非转基因玉米光合速率低于转基因玉米主要是由非气孔因素引起的,可能是参与光合作用的一些酶类受低磷胁迫的严重抑制。转基因玉米的磷浓度和磷含量较高有利于叶肉细胞内参与光合作用的酶类避免受到低磷胁迫的严重损害,因此利利于维持较高的光合作用速率。低磷胁迫严重影响了玉米雌雄穗的发育和产量的形成。但是转基因玉米受低磷影响的程度低于非转基因玉米,其单株产量显著高于非转基因玉米(P<0.05),这可能与其有较高的光合速率有关。
综上所述,在玉米中超表达TsVP可能通过促进生长素向根的运输来促进根系生长发育,而且能够通过诱导细胞膜H+-ATPase来提高玉米根际酸化能力,因此有助于提高玉米的耐低磷能力。,而目在长期低磷胁迫下转基因玉米单株产量显著高于非转基因玉米。本研究表明在玉米中过表达盐芥H-+PPase基因TsVP对提高玉米的耐低磷能力并缓解土壤缺磷造成的玉米产量损失有重要作用,为耐低磷玉米育种提供了参考资料和种质材料。
不同磷水平下耐低磷玉米自交系99038和来源亲本Qi319microRNA的差异分析
MicroRNA (miRNA)是真核生物中一类长度约为20-22个核苷酸的在转录后水平上调控基因表达的小分子非编码RNA不仅在植物的生长发育过程中起重要的调节作用,而且能够参与植物应对逆境胁迫的响应过程。越来越多的研究表明miRNA在植物低磷胁迫响应过程中起重要的调节作用，鉴定与玉米耐低磷相关的miRNA有利于深入理解玉米耐低磷机制,也能为通过基因工程的手段培育耐低磷玉米品种提供有价值的基因。本工作提供有价值的基因。本工作首次提示具有不同耐低磷能力的玉米在miRNA表达水平上的差异。所用的玉米材料为玉米自交系Qi319和其耐低磷突变体99038,99038的耐低磷能力能够稳定遗传,且保持优良的农艺性状,是鉴定玉米耐低磷相关miRNA和研究玉米耐低磷机制的优异材料。通过小RNA高通量测序技术分别在是磷条件下和在低磷条件下比较Qi319和99038根系中miRNA的表达水平,并预测miRNA候选靶基因,以期鉴定出一些可能导致玉米耐低磷能力差异的miRNA。涂了鉴定在不同磷水平下两种基因型玉米间差异表达的miRNA,通过比较同一基因型玉米低磷条件下植株与足磷条件下植株的miRNA的表达水平,以期鉴定在玉米中受低磷胁迫调控的miRNA。
将玉米培养至一叶一心时,去除胚乳,然后分成两组培养。一组在足磷(sufficient phosphate, SP,1,000μM KH2PO4)营养液中培养,另一组在低磷(low phosphate, LP,5μM KH2PO4)营养液中培养,共同培养11天。两种基因型玉米表现出不同的耐低磷能力,99038生长受低磷抑制的程度低于Qi319,其根系生物量和植株生物量都显著高于Qi319(P<005)。无论在足磷条件下还是在低磷条件下,99038的根系都比Qi319发达,其根系生物量、侧根数目、侧根长度、轴生根长度、总根长和根冠比都显著高Qi319(P<0.05)。
分别取足磷和低磷水平下培养11天的Qi319和99038的根系,提取小RNA,构建四个小RNA文库(Qi319SP,99038SP,Qi319LP和99038LP),并进行高通量测序。从Qi319SP、99038SP、Qi319LP和99038LP四个小RNA文库中分别鉴定出193、205、208和212个玉米已知miRNA,它们分别属于25、25、26和26个miRNA家族,并且分刖鉴定到46、59、50和54个新miRNA (novel miRNA)。这些新miRNA的前体长度在67到331个核苷酸之间,平均为159个核苷酸。成熟的新miRNA的长度在20到23个核苷酸之间,大多数为21个核甘酸(53%)和22个核苷酸睃(36%)。前体的最小自由能在-18.4到-128.3kcal/mol之间,平均(?)-60.59kcal/mol。
对鉴定到的26个已知miRNA家族都预洲到了靶基因。这些靶基因包括转录因子基因：SBP转录因子(miR156)、MYB转录因子(miR159、miR164和miR319)、生长素响应因子ARF转录因子(mi R160)、NAC转录因子(miR164)、 NFYA转录因子(miR169)、AP2转录因子(miR172)和GAMYB转录因子(miR319)。参与营养平衡的靶基因：铵转运体(miR162)、ATP硫酸化酶(miRNA395)、磷转运体(miR399)、铜/锌超氧化物歧化酶(miR398)和SPX结构域蛋白(miR827)基因等。参与植物生长发育的靶基因HD-ZIP蛋白(miR166)、LAC多酚氧化酶(miR397和miR528)基因等。还包括多种多样有不同功能的靶基因：磷酸酶(miR393和niR319)、抗坏血酸氧化酶(miR397)和蓝铜蛋白(miR408)基因等。对于鉴定到的86个新miRNA,只对其中56个新miRNA预测到了靶基因。这些靶基因包括：转录因子、酶类、绀构蛋白类和转运体蛋白类基因等等。对30个新miRNA没有预测到靶基因,可能是因为数据库中玉米mRNA信息还不够全面。大多数miRNA都有多个靶基因。说明miRNA通常有多种功能。有许多预测到的靶基因其编码蛋白还未得到注释。
分别在足磷条件下和在低磷条件下比较Qi319和99038根系中miRNA的表达水平,鉴定在两种基因型玉米间差异表达的miRNA.在足磷条件下,来自4个已知miRNA家族(miR160、miR164、miR397和miR528)的16个成员和5个新miRNA (mir_20、mir_129、mir_45、mir_172和mir_206)在Qi319和99038间差异表达。与在Qi319SP中相比,miR160家族成员和3个新niRNA(mir_20、 mir_172和mir_206)在99038SP中上调表达,而其它miRNA在99038SP中下调表达。在低磷条件下,Qi319和99038间差异表达的miRNA的数目多于足磷条件下差异表达的数目,来自6个已知miRNA家族(miR1432、miR160、miR169、 miR164、miR397和miR528)的19个成员和5个新miRNA (mir_99、mir_100、 mir_45、mir_172和mir_206)在Qi319LP和99038LP间差异表达。与在Qi319LP中相比,miR160家族成员和2个新miRNA (mir_172和mir_206)在99038LP文库中上调表达,而其它miRNA在99038LP文厍中下调表达。miRNA在基因型间的差异表达可能会引起两种基因型玉米耐低磷能力的差异。
除了鉴定不同供磷水平下两种基因型玉米间差异表达的miRNA,通过比较同一基因型玉米在低磷条件下的植株与在足磷条件下的植株的miRNA的表达水平,鉴定出一些在玉米中受低磷胁迫调控的miRNA来自10个已知miRNA家族的成员和6个新miRNA在Qi319和99038中受低磷胁迫调控。其中大部分玉米低磷响应的玉米已知miRNA在低磷胁迫条件下都是下调表达的,只有两个miRNA家族(miR399和miR827)的成员在低磷条件下是上调表达的,说明miRNA的下调表达在玉米低磷响应调节途径中有比较重要的作用。
通过real-time PCR确认miRNA表达水平。发现脱real-time PCR分析的结果与高通量测序结果相符合,说明测序结果比较可靠。miRNA表达模式分析结果表明,高通量测序结果显示的在低磷胁迫11天时在Qi319和99038间差异表达的一些miRNA在低磷胁迫的多个时间点(1、3、7、11和14天)在基因型间都是差异表达的(P<0.01),而且也起了某些靶基因在玉米基因型间的差异表达(P<0.01),这些靶基因包括miR160的靶基因生长素响应因子基因ARF2、miR164的靶基因MYB转录因子基因和NAC转录因子基因NAC5和miR397的靶基因laccase基因LAC4。有研究表明这些基因在玉米根系发育和MYB转录因子参与的转录调节中起重要作用。
综上所述,本工作通过小RNA高通量测序技术分别在足磷条件下和低磷条件下比较玉米自交系Qi319和其耐低磷突变体99038根系中(?)-niRNA的表达水平,发现6个玉米已知miRNA家族和7个新miRNA在两种基因型玉米间差异表达。miRNA靶基因预测以及基因表达水平分析表明某些基因型间差异表达的miRNA会引起两种基因型玉米的根系发育差异和MYB转录因子参与的转录调节差异,推测这些差异可能是99038耐低磷能力提高的原因。本工作鉴定到一些可能与提高玉米耐低磷能力有关的miRNA,为深入理解玉米耐低磷机制和发展耐低磷玉米品种提供了有价值的资料。
Phosphorous (P) is a macronutrient that is essential for plant growth, development, and reproduction. Despite the importance of P in agricultural production, most P in the soil is unavailable for plant because of the low availability of soluble phosphate (Pi), the major form of P absorbed by plant roots. Therefore, Pi availability is usually a constraint on plant productivity in many natural and agricultural ecosystems Maize (Zea mays) is an important grain and forage crop worldwide. Pi availability is critical in the early developmental stages of maize and therefore has an important effect on production. However, maize is adversely affected by Pi deficiency in many areas where it is grown, particularly in the acid soils of tropical and subtropical regions and the calcareous soils of temperate regions. These soils account for more than half the area under maize cultivation. The use of Pi-rich fertilizer can improve crop yields that are limited by Pi deficiency but this practice is costly and dangerous to aquatic ecosystems because of the resulting eutrophication. Therefore, understanding of the mechanism behind Pi tolerance in maize and development of maize crops with improved low Pi tolerance is of significance to both agriculture and the environment.
Overexpression of Thellungiella Halophila H+-pyrophosphatase gene TsVP improves low phosphate tolerance in maize
Engineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. Many studies suggested that the overexpression of vacuolar H-pyrophosphatase (H-PPase) gene could enhance abiotic stress tolerance and improve root growth in many plants. As the primary organ involved in the efficient uptake of all the mineral elements, more robust root systems can facilitate Pi uptake, it is important for improving low Pi tolerance in plants To test whether the H-PPase could improve low Pi tolerance in maize, transgenic maize plants that overexpressed1helhingiella Halophila H-PPase Gene TsVP were tested for their performance under Pi deficit stress conditions.
Maize seedlings were cultured in SP (sufficient phosphate,1,000μM KH2PO4) nutrient solution for20days, then divided into two groups and grown separately in SP and LP (low phosphate,5μM KH2PO4) nutrient solutions for an additional25days After25days of Pi deficit stress, all plants were adversely affected However, the transgenic plants displayed less growth retardation than the wild type, they showed significantly higher shoot biomass、root biomass and plant biomass than wild type (P P<0.05), suggesting that transgenic plants had enhanced low phosphate tolerance Under both SP and LP conditions, the root dry weight and root dry weight to shoot dry weight ratio in transgenic plans were also significantly higher than in the wild type (P<0.05). Moreover, transgenic plans had more axile roots, more lateral roots, longer total root length, larger root volume, larger root absorptive surface area and longer average lateral root length compared to the wild type under both SP and LP conditions. These data suggested that overexpression of TsVP in maize improved root growth significantly. There were no significant differences (P>0.05) in root soluble sugar content and root sugar content/shoot sugar content ratio between wild type and transgenic maize plants under both SP and LP conditions, suggesting that the distribution of soluble sugar in plant was not the reason for the enhanced root growth in transgenic plants The IAA content was higher in roots of transgenic plants than in roots of the wild type By contrast, the IAA content was lower in shoots of transgenic plants than in shoots of the wild type. But there was no difference in the total IAA content between the wild type and transgenic plants. Moreover, the expression levels of some genes involved in auxin transport were analyzed by real-time PCR, including ZmPINla, ZmPINlb and ZmAUX1. Transgenic maize plants showed a significantly higher (P<0.05) relative expression level of these genes compared to the wild type plants. These results suggested that overexpression of TsVP in maize might change the transport of1AA.1AA or NPA were added to wild type and transgenic plants for14days, after14days of treatment with IAA or NPA, there no significant differences (P>0.05) in root growth between wild type and transgenic plants anymore. These results suggested that IAA might be involved in the enhanced root growth in transgenic plants.
Transgenic plants showed significantly lower (P<0.05) pH value of nutrient solution than wild type under Pi deficiency, which suggested that transgenic maize plants had a enhanced rhizosphere acidification ability compared to wild type. There were no significant differences (P>0.05) in organic acid secretion rate between wild type and transgenic plants, suggesting that organic acid secretion rate was not the reason for the enhanced rhizosphere acidification ability in transgenic plants. The activity of plasmalemma H+-ATPase (P-ATPase) was significantly higher (P<0.05) in transgenic plants than in wild type under both SP and LP conditions, suggesting that overexpression of TsVP in maize significantly increased the activity of P-ATPase, which was the reason for the enhanced rhizosphere acidification ability in transgenic plants. These results indicated that the enhanced root growth and increased rhizosphere acidification ability caused by higher (P<0.05) P-ATPase might play important role in improving low phosphate tolerance in transgenic maize plants.
Transgenic maize plants had significantly higher (P<0.05) Imax values than the wild type plants, however, the Cmin and Km values in transgenic plants showed no significant differences (P>0.05) from the wild type plants under both of SP and LP conditions, which indicated that there was no difference in the affinity of Pi transporters for Pi between transgenic plants and wild type. Therefore, it could be concluded that the higher Imax values seen in transgenic plants were not due to the difference in the affinity of Pi transporters for Pi, might be due to the larger root systems in transgenic plants. Transgenic maize plants accumulated greater amount of P in shoots and roots than the wild type, this result was consistent with the higher Pi influx rate in transgenic plants.
Whether under SP and under LP conditions, APase activity in the transgenic plants showed no significant difference (P>0.05) from the wild type, which suggested that the less growth retardation under Pi deficit stress and enhanced performance displayed by transgenic plants had no correlation with the APase activity
In addition to the enhanced low Pi tolerance compared to the wild type when cultured under hydroponic conditions, maize plants that overexpressed TsFP also showed improved performance compared to the wild type plants when grown in low Pi soil. The transgenic plants showed improved root growth and shoot growth in low Pi soil compared to the wild type. The photosynthetic rates and stomatal conductance value were smaller while the intercellular CO2concentration was higher in maize grown under LP conditions compared to maize grown under SP conditions, suggesting that the decrease in photosynthetic capacity caused by low phosphate stress was mainly due to non-stomatal factors. The photosynthetic rates and stomatal conductance value were higher in transgenic plants than in wild type plants while the intercellular CO2concentration was lower in transgenic plants than in wild type plants, suggesting that the decrease in photosynthetic capacity in wild type was mainly due to non-stomatal factors Possibly because of the higher photosynthetic capacity, transgenic plants produced higher grain yield per plant than wild type plants
In summary, the results showed that the overexpression of TsVP in maize plants improved root growth and enhanced rhizosphere acidification and these phenotypes can help confer a higher degree of low phosphate tolerance to transgenic plant Most importantly, overexpression of TsVP in maize plants could increase grain yield per plant of maize under low Pi stress. This research indicated that the TsVP gene has the potential to be used for improving crop's low phosphate tolerance and yields in areas where low Pi availability is a limiting factor for agricultural productivity
Identification and comparative analysis of low phosphate tolerance-associated microRNAs in two maize genotypes
MicroRNAs (miRNAs) are known to play critical roles in plant responses to low Pi stress. The identification of low Pi tolerance-associated miRNAs can help in the selection and manipulation of high performing maize genotypes under low Pi-fertilizer conditions. Although numerous Pi starvation-responsive miRNAs have been identified in several plants, miRNAs associated with low Pi tolerance have not been identified. The comparison of miRNA expression profiles of maize genotypes differing in low Pi tolerance should provide useful information that will lead to the identification of low Pi tolerance-associated miRNAs and improve understanding of the mechanism behind low Pi tolerance in plants.
We grew two maize genotypes (wild type, Qi319, and a low-Pi tolerant mutant,99038) under SP (sufficient phosphate,1,000μM KH2PO4) and LP (low phosphate,5μM KH2PO4) conditions for11days. As expected, under LP conditions, the two maize genotypes were significantly different with regards to low Pi tolerance. The growth of the99038plants was less affected by the lack of Pi. The99038plants accumulated significantly higher root biomass and plant biomass than the Qi319plants (P<0.05). Moreover, under both of SP and LP conditions, the number of lateral roots, the lengths of the lateral root and the axile root, the total root length, and the root-shoot ratio increased significantly (P<0.05) in99038compared to the Qi319genotype.
The maize roots were collected in order to construct small RNA libraries (Qi319SP,99038SP, Qi319LP, and99038LP). The libraries were sequenced using Solexa technology. From the Qi319SP,99038SP, Qi319LP, and99038LP libraries, we identified193,205,208, and212known miRNAs belonging to25,25,26, and26miRNA families, respectively. We also identified46,59,50, and54novel miRNAs, respectively. The length of the predicted novel miRNA precursors varied from67to331nt, with an average of159nt. The length of the mature miRNAs ranged from20to23nt, the majority were21nt in length. The minimum free energy (MFE) values of precursors were in the range:-18.4to-128.3kcal mol-1, with an average of-60.59kcal mol-1. We were able to predict targets for26known maize miRNA families. These targets included transcription factors, such as SBP (miR156), MYB (miR159, miR164, miR319), ARF (miR.160), NAC (miR164), NFYA (miR169), AP2-type transcription factors (miR172), and GAMYB (miR319). Other miRNA targets include genes involved in nutrient homeostasis, such as ammonium transporters (miR162), ATP sulfurylase (miRNA395), phosphate transporters (miR399), Cu/Zn superoxide dismutase CSDs (miR398), SPX domain proteins (miR827), and genes implicated in plant development, such as HD-ZIP proteins (miR166) and laccases (miR397, miR528), and other genes with diverse functions, such as phosphatase (miR393, miR319), ascorbate oxidase (miR397), blue copper protein (miR408), and50S ribosomal protein (miR167) In many cases, some targets were identified as uncharacterized protein. The target genes for56novel miRNAs were successfully predicted. The novel miRNAs from maize target a variety of genes involved in diverse biological functions, including transcription factors, various enzymes. structural proteins, and transport proteins. Most miRNAs had multiple target sites, which suggested that these miRNAs have different functions. In most cases the targets were identified as uncharacterized protein However, for30novel miRNAs, we failed to discover any targets for them in maize As complete annotations for the sequence information in maize become more available, then more accurate target prediction and verification will be possible.
In order to identify the differentially expressed miRNAs between Qi3l9and99038, the expression level of miRNAs between the two maize genotypes were compared under SP and LP conditions. Under SP conditions, members of four known miRNA families (miR160, miR164, miR.397, and miR528) and five novel miRNAs (mir20, mir_129, mir_45, mir_172, and mir_206) were differentially expressed between Qi319and99038. Among them, members of the miRI60family and three novel miRNAs (mir20, mir_172, and mir_206) were up-regulated, whereas the others were down-regulated in99038SP compared to Qi319SP Three novel miRNAs (mir_20, mir_172, and mir206) were only expressed in99038SP, whereas the other two (mir_45and mir_129) were only expressed in Qi3l9SP. Under LP conditions, the number of differently expressed miRNAs between the genotypes was greater than that under SP conditions. Overall, under LP conditions, members from six known miRNA families (miR1432, miR160, miR169, miR164, miR397, and miR528) and five novel miRNAs (mir_99, mir_100, mir45, mir_172, and mir_206) were differentially expressed between Qi319and99038. Members of four known miRNA families (miR160, miR164, miR397, and miR528) and three novel miRNAs (mir_45, mir_172, and mir206) were differently expressed between genotypes, regardless of the treatments. However, miR1432, miR169a-3p, miR169b-3p, and two novel miRNAs (mir_99and mir_100) were found to be differentially expressed between Qi319and99038under the LP conditions only and mir20and mir_129were differentially expressed between Qi319and99038under the SP conditions only.
In order to identify the Pi starvation responsive miRNAs, miRNA expression levels between low Pi stressed samples and their corresponding control samples for each genotype were compared. Ten known miRNA families and six novel miRNAs were significantly regulated by low Pi stress in both Qi319and99038. Most of Pi starvation responsive miRNAs were down-regulated by low Pi stress and only two known miRNA families (miR399and miR827) were up-regulated, which suggested that down-regulation of miRNAs appeared to be more important in low Pi response However, five novel miRNAs (mir_129, mir_20, mir_45, mir_99, and mir_100) were regulated by low Pi stress in only Qi319and one (mir_206) in only99038.
Eight of the miRNAs that were differentially expressed between the genotypes and nine low Pi responsive miRNAs were selected for validation of their expression levels by real-time PCR. The expression analysis showed a good consistency between the results derived from the high-throughput sequencing and the real-time PCR results.
In order to study the expression pattern of miRNAs at different stages of low Pi stress, real-time PCR was performed on the leaves and roots of Qi319and99038at different time points (1,3,7,11, and14days) after low Pi stress for six miRNAs (miR160, miR164, miR397, miR398, miR399, and novel miRNA, mir_206). The99038genotype showed significantly higher (P<0.01) expression levels of miR160and mir_206and lower (P<0.01) expression levels of miR164and miR397in roots than Qi319at several time points (1,3,7,11, and14days) after low Pi stress. The expression profiles of some predicted target genes were analyzed by real-time PCR. These targets included auxin response factor gene ARh'2targeted by miR160, MYB transcription factor and NAC domain transcription factor gene NAC5targeted by miR.164and laccase gene LAC4targeted by miR397. The expression levels of these target genes were also significantly different (P<0.01) between two maize genotype roots at several time points (1,3,7,11, and14days) after low Pi stress These results suggested that the differentially expressed miRNAs between genotypes identified by the sequencing analysis at11days after low Pi stress, were also differently expressed between genotypes at other growth stages and caused different expression of some of their target genes between genotypes Many studies suggested that these target genes are involved in root development in plants or play important roles in the transcription regulation of Pi starvation responses in plants.
In summary, we compared miRNA expression levels in a low Pi tolerant mutant and a wild type maize genotype under different Pi conditions by deep sequencing technology. This led to the identification of six known miRNA families and seven novel miRNAs that were differently expressed between genotypes By comparing the expression level of miRNAs in the low Pi stressed samples to the control samples, we also identified10known miRNA families and12novel miRNAs that were responsive to Pi starvation in maize. Subsequent target gene prediction and expression analysis suggested that differently expressed miRNAs might play important roles in root development and transcription regulation of Pi starvation responses, which might contribute to different levels of low Pi tolerance in different maize genotypes. This study identified some miRNAs might help in improving low Pi tolerance in maize, leading to better understanding of the mechanism behind low Pi tolerance in maize.
转TsVP提高玉米低磷耐受性的研究
通过植物基因工程的手段将有价值的基因转入玉米是发展耐低磷玉米品种的重要途径。许多研究表明超表达氢离子焦磷酸酶(H+-PPase)基因能够在多种植物中促进植物根系生长发育并能提高植物的抗逆性。在高等植物中,根系是植物吸收矿质营养的主要器官,尤其磷素在土壤中的移动性很低,所以根系的生长发育和形态结构对捕获吸收土壤磷素和提高植物耐低磷能力尤为重要。因此为了探索H+-PPase能否提高玉米的耐低磷能力,本工作中我们选用玉米自交系DH4866以及其转盐芥H+-PPase基因(TsVP)玉米为实验材料,比较两者的耐低磷能力以及在低磷条件下的产量,以期得到耐低磷能力提高的转基因玉米材料,为培养耐低磷玉米品种提供育种材料。
将玉米在足磷(sufficient phosphate, SP,1,000μM KH2PO4)营养液中培养20天,然后一半植株在足磷营养液中培养,另一半株在低磷(low phosphate, LP,5μM KH2PO4)营养液中培养,共同培养25天。低磷条件下,转基因玉米生长发育受抑制的程度低于非转基因植株,其地上部分生物量、根系生量和植株生物量都显著高于非转基因植株(P<0.05),说明转TsVP玉米有较高的耐低磷能力。无论在正常生长条件下还是低磷胁迫条件下,转基因玉米的根系都比非转基因植株发达,其根系生物量干重、根冠比、侧根数目、侧根长度、总根长和根系吸收面积都显著高于非转基因植株(P<005),说明在玉米中超表达TsVP能够显著促进玉米根系生长发育。转基因玉米的茎叶和根部可溶性总糖含量和可溶性总糖含量根冠比与非转基因植株相比都没有显著差异(P>0.05),说明碳水化合物的含量和分配不是造成转基因玉米和非转基因玉米根系差异的原因。无论在足磷条件下还是在低磷条件下,转基因玉米植株生长素总量非转基因植株相比无差别,但是根系生长素含量明显高于非转基因植株,而茎叶生长素含量明显低于非转基因植株,说明在玉米中超表达TsFP能够促进生长素向根系分配。转基因玉米中几个生长素运输相关基因(ZmAU.X1、ZmPIN1a和ZmPIN1b)的表达水平显著高于非转基因植株(P<005),说明过表达TsVP可能影响了玉米植株体内生长素运输过程。对转基因玉米和非转基因玉米都外源施加IAA或NPA后,它们在根系生物量和根系形态参数方面的显著性差异消失。这些结果都说明转基因玉米和非转基因玉米之间在根系方面的差异涉及生长素的参与,说明在玉米中超表达TsVP可能是通过促进生长素向根部运输从而促进转基因玉米根系发达。
不论在足磷条件下还是低磷条件下,转基因玉米的最大吸收速率Imax值都显著高于非转基因植株(P<005),但是(min和Km值与非转基因植株相比无显著差异(P>0.05),说明玉米磷转运体对磷的亲和力不是导致转基因玉米与非转基因玉米磷吸收速率差异的原因,因此推测转基因玉米有较高的吸收速率与其有较发达根系系统有关。转基因玉米茎叶和磷含量都显著高于非转基因植株(P<0.05),这与其有较高的磷吸收速率相一致。较高的磷浓度对低磷胁迫下的植株的生长发育极有利,这可能是低磷条件下转基因玉米生长发育受低磷抑制的程度较低的原因。
转基因玉米培养液的pH值显著低于非转基因植株(P<005),表明其根际酸化能力高于非转基因植株。但是其根系有机酸分泌速率与和非转基因玉米相比无显著差异(P>0.05),说明有机酸分泌速率并不是转基因玉米有较强根际酸化能力的原因。但昌无论在是磷条件还是在低磷条件下,转基因玉米的P*ATPase酶活性都显著高于非转基因植株(P<005),说明在米中超表达TsVP能够通过提高P*ATPase(?)活性从面提高玉米根际酸化能力。
无论在足磷条件下还是低磷条件下转基因玉米和非转基因植株相应部位的组织内酸性磷酸酶活性外泌酸性磷酸酶的活性均没有显著差异(P>005)。说明超表达TsVP并没有改变玉米的酸性磷酸酶活性,其并不是转基因玉米耐低磷能力提高的原因。
以上结果表明在玉米中超表达盐芥H-PPase基因TsVP可能通过促进生长素向根中运输来促进玉米根系生长发育而且能够能过诱导细胞膜H-ATPase来提高玉米根际酸化能力。,这些特征都与植物适应低磷胁迫相关,根系发达以及根际酸化能力增强都有利于植物在低磷条件下的生长。因此推测较发达的根系系统和较高的根际酸化能力是转TsVP玉米耐低磷能力提高的原因。
在低磷土壤中,转基因玉米在不同时时期的生长发育受低磷抑制的程度均低于非转基因玉米,其茎叶生物理、根系生物量、植株生物量和生物量的根冠比在各个生长阶段都显著高于非转基因玉米(P<0.05),说明在玉米中超表达TsVP在各个生长阶段都能促进玉米根系的生长发育和提高玉米的耐低磷能力。在低磷土壤中生长的转基因玉米的磷浓度和磷含量显著高于非转基因玉米(P<0.05),这与其有较发达的根系和较高的根际酸化能力相一致,而较高的磷浓度又有利于植物在低磷条件下生长发育,这可能是低磷土壤中转基因玉米生长发育受低磷胁迫抑制的程度低于非转基因玉米的原因。
在低磷土壤中生长的玉米的净光合值和气孔导度明显显低于在足磷土壤中生长的玉米,而胞间二氧化碳浓度明显高于在足磷土壤中生长的玉米,说明低磷胁迫造成的植物光合速率下降主要是由非气孔因素引起的。在低磷土壤中,转基因玉米的净光合值和气孔导度暄著高于非转基因植株(P<0.05),而胞间二氧化碳浓度显著低于非转基因植株(P<0.05),说明非转基因玉米光合速率低于转基因玉米主要是由非气孔因素引起的,可能是参与光合作用的一些酶类受低磷胁迫的严重抑制。转基因玉米的磷浓度和磷含量较高有利于叶肉细胞内参与光合作用的酶类避免受到低磷胁迫的严重损害,因此利利于维持较高的光合作用速率。低磷胁迫严重影响了玉米雌雄穗的发育和产量的形成。但是转基因玉米受低磷影响的程度低于非转基因玉米,其单株产量显著高于非转基因玉米(P<0.05),这可能与其有较高的光合速率有关。
综上所述,在玉米中超表达TsVP可能通过促进生长素向根的运输来促进根系生长发育,而且能够通过诱导细胞膜H+-ATPase来提高玉米根际酸化能力,因此有助于提高玉米的耐低磷能力。,而目在长期低磷胁迫下转基因玉米单株产量显著高于非转基因玉米。本研究表明在玉米中过表达盐芥H-+PPase基因TsVP对提高玉米的耐低磷能力并缓解土壤缺磷造成的玉米产量损失有重要作用,为耐低磷玉米育种提供了参考资料和种质材料。
不同磷水平下耐低磷玉米自交系99038和来源亲本Qi319microRNA的差异分析
MicroRNA (miRNA)是真核生物中一类长度约为20-22个核苷酸的在转录后水平上调控基因表达的小分子非编码RNA不仅在植物的生长发育过程中起重要的调节作用,而且能够参与植物应对逆境胁迫的响应过程。越来越多的研究表明miRNA在植物低磷胁迫响应过程中起重要的调节作用，鉴定与玉米耐低磷相关的miRNA有利于深入理解玉米耐低磷机制,也能为通过基因工程的手段培育耐低磷玉米品种提供有价值的基因。本工作提供有价值的基因。本工作首次提示具有不同耐低磷能力的玉米在miRNA表达水平上的差异。所用的玉米材料为玉米自交系Qi319和其耐低磷突变体99038,99038的耐低磷能力能够稳定遗传,且保持优良的农艺性状,是鉴定玉米耐低磷相关miRNA和研究玉米耐低磷机制的优异材料。通过小RNA高通量测序技术分别在是磷条件下和在低磷条件下比较Qi319和99038根系中miRNA的表达水平,并预测miRNA候选靶基因,以期鉴定出一些可能导致玉米耐低磷能力差异的miRNA。涂了鉴定在不同磷水平下两种基因型玉米间差异表达的miRNA,通过比较同一基因型玉米低磷条件下植株与足磷条件下植株的miRNA的表达水平,以期鉴定在玉米中受低磷胁迫调控的miRNA。
将玉米培养至一叶一心时,去除胚乳,然后分成两组培养。一组在足磷(sufficient phosphate, SP,1,000μM KH2PO4)营养液中培养,另一组在低磷(low phosphate, LP,5μM KH2PO4)营养液中培养,共同培养11天。两种基因型玉米表现出不同的耐低磷能力,99038生长受低磷抑制的程度低于Qi319,其根系生物量和植株生物量都显著高于Qi319(P<005)。无论在足磷条件下还是在低磷条件下,99038的根系都比Qi319发达,其根系生物量、侧根数目、侧根长度、轴生根长度、总根长和根冠比都显著高Qi319(P<0.05)。
分别取足磷和低磷水平下培养11天的Qi319和99038的根系,提取小RNA,构建四个小RNA文库(Qi319SP,99038SP,Qi319LP和99038LP),并进行高通量测序。从Qi319SP、99038SP、Qi319LP和99038LP四个小RNA文库中分别鉴定出193、205、208和212个玉米已知miRNA,它们分别属于25、25、26和26个miRNA家族,并且分刖鉴定到46、59、50和54个新miRNA (novel miRNA)。这些新miRNA的前体长度在67到331个核苷酸之间,平均为159个核苷酸。成熟的新miRNA的长度在20到23个核苷酸之间,大多数为21个核甘酸(53%)和22个核苷酸睃(36%)。前体的最小自由能在-18.4到-128.3kcal/mol之间,平均(?)-60.59kcal/mol。
对鉴定到的26个已知miRNA家族都预洲到了靶基因。这些靶基因包括转录因子基因：SBP转录因子(miR156)、MYB转录因子(miR159、miR164和miR319)、生长素响应因子ARF转录因子(mi R160)、NAC转录因子(miR164)、 NFYA转录因子(miR169)、AP2转录因子(miR172)和GAMYB转录因子(miR319)。参与营养平衡的靶基因：铵转运体(miR162)、ATP硫酸化酶(miRNA395)、磷转运体(miR399)、铜/锌超氧化物歧化酶(miR398)和SPX结构域蛋白(miR827)基因等。参与植物生长发育的靶基因HD-ZIP蛋白(miR166)、LAC多酚氧化酶(miR397和miR528)基因等。还包括多种多样有不同功能的靶基因：磷酸酶(miR393和niR319)、抗坏血酸氧化酶(miR397)和蓝铜蛋白(miR408)基因等。对于鉴定到的86个新miRNA,只对其中56个新miRNA预测到了靶基因。这些靶基因包括：转录因子、酶类、绀构蛋白类和转运体蛋白类基因等等。对30个新miRNA没有预测到靶基因,可能是因为数据库中玉米mRNA信息还不够全面。大多数miRNA都有多个靶基因。说明miRNA通常有多种功能。有许多预测到的靶基因其编码蛋白还未得到注释。
分别在足磷条件下和在低磷条件下比较Qi319和99038根系中miRNA的表达水平,鉴定在两种基因型玉米间差异表达的miRNA.在足磷条件下,来自4个已知miRNA家族(miR160、miR164、miR397和miR528)的16个成员和5个新miRNA (mir_20、mir_129、mir_45、mir_172和mir_206)在Qi319和99038间差异表达。与在Qi319SP中相比,miR160家族成员和3个新niRNA(mir_20、 mir_172和mir_206)在99038SP中上调表达,而其它miRNA在99038SP中下调表达。在低磷条件下,Qi319和99038间差异表达的miRNA的数目多于足磷条件下差异表达的数目,来自6个已知miRNA家族(miR1432、miR160、miR169、 miR164、miR397和miR528)的19个成员和5个新miRNA (mir_99、mir_100、 mir_45、mir_172和mir_206)在Qi319LP和99038LP间差异表达。与在Qi319LP中相比,miR160家族成员和2个新miRNA (mir_172和mir_206)在99038LP文库中上调表达,而其它miRNA在99038LP文厍中下调表达。miRNA在基因型间的差异表达可能会引起两种基因型玉米耐低磷能力的差异。
除了鉴定不同供磷水平下两种基因型玉米间差异表达的miRNA,通过比较同一基因型玉米在低磷条件下的植株与在足磷条件下的植株的miRNA的表达水平,鉴定出一些在玉米中受低磷胁迫调控的miRNA来自10个已知miRNA家族的成员和6个新miRNA在Qi319和99038中受低磷胁迫调控。其中大部分玉米低磷响应的玉米已知miRNA在低磷胁迫条件下都是下调表达的,只有两个miRNA家族(miR399和miR827)的成员在低磷条件下是上调表达的,说明miRNA的下调表达在玉米低磷响应调节途径中有比较重要的作用。
通过real-time PCR确认miRNA表达水平。发现脱real-time PCR分析的结果与高通量测序结果相符合,说明测序结果比较可靠。miRNA表达模式分析结果表明,高通量测序结果显示的在低磷胁迫11天时在Qi319和99038间差异表达的一些miRNA在低磷胁迫的多个时间点(1、3、7、11和14天)在基因型间都是差异表达的(P<0.01),而且也起了某些靶基因在玉米基因型间的差异表达(P<0.01),这些靶基因包括miR160的靶基因生长素响应因子基因ARF2、miR164的靶基因MYB转录因子基因和NAC转录因子基因NAC5和miR397的靶基因laccase基因LAC4。有研究表明这些基因在玉米根系发育和MYB转录因子参与的转录调节中起重要作用。
综上所述,本工作通过小RNA高通量测序技术分别在足磷条件下和低磷条件下比较玉米自交系Qi319和其耐低磷突变体99038根系中(?)-niRNA的表达水平,发现6个玉米已知miRNA家族和7个新miRNA在两种基因型玉米间差异表达。miRNA靶基因预测以及基因表达水平分析表明某些基因型间差异表达的miRNA会引起两种基因型玉米的根系发育差异和MYB转录因子参与的转录调节差异,推测这些差异可能是99038耐低磷能力提高的原因。本工作鉴定到一些可能与提高玉米耐低磷能力有关的miRNA,为深入理解玉米耐低磷机制和发展耐低磷玉米品种提供了有价值的资料。
Phosphorous (P) is a macronutrient that is essential for plant growth, development, and reproduction. Despite the importance of P in agricultural production, most P in the soil is unavailable for plant because of the low availability of soluble phosphate (Pi), the major form of P absorbed by plant roots. Therefore, Pi availability is usually a constraint on plant productivity in many natural and agricultural ecosystems Maize (Zea mays) is an important grain and forage crop worldwide. Pi availability is critical in the early developmental stages of maize and therefore has an important effect on production. However, maize is adversely affected by Pi deficiency in many areas where it is grown, particularly in the acid soils of tropical and subtropical regions and the calcareous soils of temperate regions. These soils account for more than half the area under maize cultivation. The use of Pi-rich fertilizer can improve crop yields that are limited by Pi deficiency but this practice is costly and dangerous to aquatic ecosystems because of the resulting eutrophication. Therefore, understanding of the mechanism behind Pi tolerance in maize and development of maize crops with improved low Pi tolerance is of significance to both agriculture and the environment.
Overexpression of Thellungiella Halophila H+-pyrophosphatase gene TsVP improves low phosphate tolerance in maize
Engineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. Many studies suggested that the overexpression of vacuolar H-pyrophosphatase (H-PPase) gene could enhance abiotic stress tolerance and improve root growth in many plants. As the primary organ involved in the efficient uptake of all the mineral elements, more robust root systems can facilitate Pi uptake, it is important for improving low Pi tolerance in plants To test whether the H-PPase could improve low Pi tolerance in maize, transgenic maize plants that overexpressed1helhingiella Halophila H-PPase Gene TsVP were tested for their performance under Pi deficit stress conditions.
Maize seedlings were cultured in SP (sufficient phosphate,1,000μM KH2PO4) nutrient solution for20days, then divided into two groups and grown separately in SP and LP (low phosphate,5μM KH2PO4) nutrient solutions for an additional25days After25days of Pi deficit stress, all plants were adversely affected However, the transgenic plants displayed less growth retardation than the wild type, they showed significantly higher shoot biomass、root biomass and plant biomass than wild type (P P<0.05), suggesting that transgenic plants had enhanced low phosphate tolerance Under both SP and LP conditions, the root dry weight and root dry weight to shoot dry weight ratio in transgenic plans were also significantly higher than in the wild type (P<0.05). Moreover, transgenic plans had more axile roots, more lateral roots, longer total root length, larger root volume, larger root absorptive surface area and longer average lateral root length compared to the wild type under both SP and LP conditions. These data suggested that overexpression of TsVP in maize improved root growth significantly. There were no significant differences (P>0.05) in root soluble sugar content and root sugar content/shoot sugar content ratio between wild type and transgenic maize plants under both SP and LP conditions, suggesting that the distribution of soluble sugar in plant was not the reason for the enhanced root growth in transgenic plants The IAA content was higher in roots of transgenic plants than in roots of the wild type By contrast, the IAA content was lower in shoots of transgenic plants than in shoots of the wild type. But there was no difference in the total IAA content between the wild type and transgenic plants. Moreover, the expression levels of some genes involved in auxin transport were analyzed by real-time PCR, including ZmPINla, ZmPINlb and ZmAUX1. Transgenic maize plants showed a significantly higher (P<0.05) relative expression level of these genes compared to the wild type plants. These results suggested that overexpression of TsVP in maize might change the transport of1AA.1AA or NPA were added to wild type and transgenic plants for14days, after14days of treatment with IAA or NPA, there no significant differences (P>0.05) in root growth between wild type and transgenic plants anymore. These results suggested that IAA might be involved in the enhanced root growth in transgenic plants.
Transgenic plants showed significantly lower (P<0.05) pH value of nutrient solution than wild type under Pi deficiency, which suggested that transgenic maize plants had a enhanced rhizosphere acidification ability compared to wild type. There were no significant differences (P>0.05) in organic acid secretion rate between wild type and transgenic plants, suggesting that organic acid secretion rate was not the reason for the enhanced rhizosphere acidification ability in transgenic plants. The activity of plasmalemma H+-ATPase (P-ATPase) was significantly higher (P<0.05) in transgenic plants than in wild type under both SP and LP conditions, suggesting that overexpression of TsVP in maize significantly increased the activity of P-ATPase, which was the reason for the enhanced rhizosphere acidification ability in transgenic plants. These results indicated that the enhanced root growth and increased rhizosphere acidification ability caused by higher (P<0.05) P-ATPase might play important role in improving low phosphate tolerance in transgenic maize plants.
Transgenic maize plants had significantly higher (P<0.05) Imax values than the wild type plants, however, the Cmin and Km values in transgenic plants showed no significant differences (P>0.05) from the wild type plants under both of SP and LP conditions, which indicated that there was no difference in the affinity of Pi transporters for Pi between transgenic plants and wild type. Therefore, it could be concluded that the higher Imax values seen in transgenic plants were not due to the difference in the affinity of Pi transporters for Pi, might be due to the larger root systems in transgenic plants. Transgenic maize plants accumulated greater amount of P in shoots and roots than the wild type, this result was consistent with the higher Pi influx rate in transgenic plants.
Whether under SP and under LP conditions, APase activity in the transgenic plants showed no significant difference (P>0.05) from the wild type, which suggested that the less growth retardation under Pi deficit stress and enhanced performance displayed by transgenic plants had no correlation with the APase activity
In addition to the enhanced low Pi tolerance compared to the wild type when cultured under hydroponic conditions, maize plants that overexpressed TsFP also showed improved performance compared to the wild type plants when grown in low Pi soil. The transgenic plants showed improved root growth and shoot growth in low Pi soil compared to the wild type. The photosynthetic rates and stomatal conductance value were smaller while the intercellular CO2concentration was higher in maize grown under LP conditions compared to maize grown under SP conditions, suggesting that the decrease in photosynthetic capacity caused by low phosphate stress was mainly due to non-stomatal factors. The photosynthetic rates and stomatal conductance value were higher in transgenic plants than in wild type plants while the intercellular CO2concentration was lower in transgenic plants than in wild type plants, suggesting that the decrease in photosynthetic capacity in wild type was mainly due to non-stomatal factors Possibly because of the higher photosynthetic capacity, transgenic plants produced higher grain yield per plant than wild type plants
In summary, the results showed that the overexpression of TsVP in maize plants improved root growth and enhanced rhizosphere acidification and these phenotypes can help confer a higher degree of low phosphate tolerance to transgenic plant Most importantly, overexpression of TsVP in maize plants could increase grain yield per plant of maize under low Pi stress. This research indicated that the TsVP gene has the potential to be used for improving crop's low phosphate tolerance and yields in areas where low Pi availability is a limiting factor for agricultural productivity
Identification and comparative analysis of low phosphate tolerance-associated microRNAs in two maize genotypes
MicroRNAs (miRNAs) are known to play critical roles in plant responses to low Pi stress. The identification of low Pi tolerance-associated miRNAs can help in the selection and manipulation of high performing maize genotypes under low Pi-fertilizer conditions. Although numerous Pi starvation-responsive miRNAs have been identified in several plants, miRNAs associated with low Pi tolerance have not been identified. The comparison of miRNA expression profiles of maize genotypes differing in low Pi tolerance should provide useful information that will lead to the identification of low Pi tolerance-associated miRNAs and improve understanding of the mechanism behind low Pi tolerance in plants.
We grew two maize genotypes (wild type, Qi319, and a low-Pi tolerant mutant,99038) under SP (sufficient phosphate,1,000μM KH2PO4) and LP (low phosphate,5μM KH2PO4) conditions for11days. As expected, under LP conditions, the two maize genotypes were significantly different with regards to low Pi tolerance. The growth of the99038plants was less affected by the lack of Pi. The99038plants accumulated significantly higher root biomass and plant biomass than the Qi319plants (P<0.05). Moreover, under both of SP and LP conditions, the number of lateral roots, the lengths of the lateral root and the axile root, the total root length, and the root-shoot ratio increased significantly (P<0.05) in99038compared to the Qi319genotype.
The maize roots were collected in order to construct small RNA libraries (Qi319SP,99038SP, Qi319LP, and99038LP). The libraries were sequenced using Solexa technology. From the Qi319SP,99038SP, Qi319LP, and99038LP libraries, we identified193,205,208, and212known miRNAs belonging to25,25,26, and26miRNA families, respectively. We also identified46,59,50, and54novel miRNAs, respectively. The length of the predicted novel miRNA precursors varied from67to331nt, with an average of159nt. The length of the mature miRNAs ranged from20to23nt, the majority were21nt in length. The minimum free energy (MFE) values of precursors were in the range:-18.4to-128.3kcal mol-1, with an average of-60.59kcal mol-1. We were able to predict targets for26known maize miRNA families. These targets included transcription factors, such as SBP (miR156), MYB (miR159, miR164, miR319), ARF (miR.160), NAC (miR164), NFYA (miR169), AP2-type transcription factors (miR172), and GAMYB (miR319). Other miRNA targets include genes involved in nutrient homeostasis, such as ammonium transporters (miR162), ATP sulfurylase (miRNA395), phosphate transporters (miR399), Cu/Zn superoxide dismutase CSDs (miR398), SPX domain proteins (miR827), and genes implicated in plant development, such as HD-ZIP proteins (miR166) and laccases (miR397, miR528), and other genes with diverse functions, such as phosphatase (miR393, miR319), ascorbate oxidase (miR397), blue copper protein (miR408), and50S ribosomal protein (miR167) In many cases, some targets were identified as uncharacterized protein. The target genes for56novel miRNAs were successfully predicted. The novel miRNAs from maize target a variety of genes involved in diverse biological functions, including transcription factors, various enzymes. structural proteins, and transport proteins. Most miRNAs had multiple target sites, which suggested that these miRNAs have different functions. In most cases the targets were identified as uncharacterized protein However, for30novel miRNAs, we failed to discover any targets for them in maize As complete annotations for the sequence information in maize become more available, then more accurate target prediction and verification will be possible.
In order to identify the differentially expressed miRNAs between Qi3l9and99038, the expression level of miRNAs between the two maize genotypes were compared under SP and LP conditions. Under SP conditions, members of four known miRNA families (miR160, miR164, miR.397, and miR528) and five novel miRNAs (mir20, mir_129, mir_45, mir_172, and mir_206) were differentially expressed between Qi319and99038. Among them, members of the miRI60family and three novel miRNAs (mir20, mir_172, and mir_206) were up-regulated, whereas the others were down-regulated in99038SP compared to Qi319SP Three novel miRNAs (mir_20, mir_172, and mir206) were only expressed in99038SP, whereas the other two (mir_45and mir_129) were only expressed in Qi3l9SP. Under LP conditions, the number of differently expressed miRNAs between the genotypes was greater than that under SP conditions. Overall, under LP conditions, members from six known miRNA families (miR1432, miR160, miR169, miR164, miR397, and miR528) and five novel miRNAs (mir_99, mir_100, mir45, mir_172, and mir_206) were differentially expressed between Qi319and99038. Members of four known miRNA families (miR160, miR164, miR397, and miR528) and three novel miRNAs (mir_45, mir_172, and mir206) were differently expressed between genotypes, regardless of the treatments. However, miR1432, miR169a-3p, miR169b-3p, and two novel miRNAs (mir_99and mir_100) were found to be differentially expressed between Qi319and99038under the LP conditions only and mir20and mir_129were differentially expressed between Qi319and99038under the SP conditions only.
In order to identify the Pi starvation responsive miRNAs, miRNA expression levels between low Pi stressed samples and their corresponding control samples for each genotype were compared. Ten known miRNA families and six novel miRNAs were significantly regulated by low Pi stress in both Qi319and99038. Most of Pi starvation responsive miRNAs were down-regulated by low Pi stress and only two known miRNA families (miR399and miR827) were up-regulated, which suggested that down-regulation of miRNAs appeared to be more important in low Pi response However, five novel miRNAs (mir_129, mir_20, mir_45, mir_99, and mir_100) were regulated by low Pi stress in only Qi319and one (mir_206) in only99038.
Eight of the miRNAs that were differentially expressed between the genotypes and nine low Pi responsive miRNAs were selected for validation of their expression levels by real-time PCR. The expression analysis showed a good consistency between the results derived from the high-throughput sequencing and the real-time PCR results.
In order to study the expression pattern of miRNAs at different stages of low Pi stress, real-time PCR was performed on the leaves and roots of Qi319and99038at different time points (1,3,7,11, and14days) after low Pi stress for six miRNAs (miR160, miR164, miR397, miR398, miR399, and novel miRNA, mir_206). The99038genotype showed significantly higher (P<0.01) expression levels of miR160and mir_206and lower (P<0.01) expression levels of miR164and miR397in roots than Qi319at several time points (1,3,7,11, and14days) after low Pi stress. The expression profiles of some predicted target genes were analyzed by real-time PCR. These targets included auxin response factor gene ARh'2targeted by miR160, MYB transcription factor and NAC domain transcription factor gene NAC5targeted by miR.164and laccase gene LAC4targeted by miR397. The expression levels of these target genes were also significantly different (P<0.01) between two maize genotype roots at several time points (1,3,7,11, and14days) after low Pi stress These results suggested that the differentially expressed miRNAs between genotypes identified by the sequencing analysis at11days after low Pi stress, were also differently expressed between genotypes at other growth stages and caused different expression of some of their target genes between genotypes Many studies suggested that these target genes are involved in root development in plants or play important roles in the transcription regulation of Pi starvation responses in plants.
In summary, we compared miRNA expression levels in a low Pi tolerant mutant and a wild type maize genotype under different Pi conditions by deep sequencing technology. This led to the identification of six known miRNA families and seven novel miRNAs that were differently expressed between genotypes By comparing the expression level of miRNAs in the low Pi stressed samples to the control samples, we also identified10known miRNA families and12novel miRNAs that were responsive to Pi starvation in maize. Subsequent target gene prediction and expression analysis suggested that differently expressed miRNAs might play important roles in root development and transcription regulation of Pi starvation responses, which might contribute to different levels of low Pi tolerance in different maize genotypes. This study identified some miRNAs might help in improving low Pi tolerance in maize, leading to better understanding of the mechanism behind low Pi tolerance in maize.
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