干旱胁迫下油菜抗倒伏相关性状动态变化及木质素关键基因表达特性分析
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摘要
我国食用植物油自给率不到40%,是我国大宗农产品中安全形势最严峻的作物,制约我国油菜生产面积扩展的主要限制因素为油菜抗性弱和机械化水平低。油菜倒伏不仅产量比正常油菜减产10%～30%,严重的可达50%以上(姜维梅等,2001),而且品质差,含油量亦比正常油菜低10%～30%,也是油菜机械收割的限制性因素。旱害也是我国最常见、对农业生产影响最大的气候灾害,旱灾面积占农作物总受灾面积的一半以上,严重干旱年份比例高达75%(朱宗河,2011)。因此,研究干旱胁迫下油菜茎秆木质素基因的表达调控机制,可为提高油菜抗旱、抗倒能力,选育广适、多抗、耐机播机收油菜新品种提供理论依据。
本研究以10份较稳定的抗倒伏和易倒伏极端表现型甘蓝型油菜品种(系)为材料,通过旱棚内干旱胁迫和正常水分对照的对比试验,分析了抗倒伏与易倒伏极端表现型材料的茎秆相关性状、茎杆木质素含量及木质素单体比例等性状在不同发育时期的动态变化规律,比较了不同发育时期茎秆中木质素合成相关基因的表达差异,初步揭示了干旱胁迫下甘蓝型油菜抗倒伏特性形成的机理。主要研究结果如下：
1)干旱胁迫下油菜抗倒伏相关性状与茎杆抗折力间的关系。在油菜易发生倒伏的四个时期(蕾苔期、初花期、终花期、青荚期),每个材料在干旱组和对照组中各取3株,调查了株高、茎杆中部直径、含水量、抗折断力等性状,详细分析了各性状在两种环境下的动态变化差异；采用通径分析方法分析了株高、茎杆中部直径、含水量对茎杆抗折断力的影响。结果发现：(1)四个性状在干旱胁迫下的表现均与正常环境有明显差别,一般趋势是,干旱胁迫环境下,油菜植株变矮、茎杆含水量减少、茎杆直径变小但抗折断力增加；(2)各参试材料除受环境影响有共同的变化趋势以外,在部分发育时期有明显的基因型-环境互作现象存在；(3)通径分析表明在两种环境下,油菜的株高对茎秆的抗折力有最大的直接作用,直径对茎秆的抗折力也有一定的直接决定作用,剩余通径系数表明除株高和直径外还有其他一些因素决定茎秆的抗折能力。
2)干旱胁迫下油菜茎杆木质素含量、单体比率与油菜茎杆抗折性关系。在油菜易发生倒伏的四个时期,每个材料在于旱组和对照组中各取3株,测定了中部茎秆的木质素含量和木质素单体比例。结果表明：(1)终花期至青荚期间是油菜茎秆木质素的快速积累期；(2)在不同时期和不同环境处理下,茎杆木质素含量受环境影响很大,除蕾苔期外茎杆木质素在干旱条件下合成积累加快,其余时期茎杆木质素的合成速率还与基因型有关,存在明显的基因型-环境互作现象；(3)木质素含量与抗折性间的相关分析表明,两种环境条件下,油菜茎秆木质素的含量与茎秆抗折性间均存在显著或极显著的正相关关系,说明茎杆木质素含量愈高,茎秆的机械强度愈强,抗折性也愈强；(4)油菜H型木质素平均仅占总木质素总成分的10.96%,且各材料间相差不大；油菜木质素单体主要为S-G型木质素,各材料间木质素单体S/G比率有极显著差异；(5)蕾薹期至青荚期,两种环境下参试材料的主要木质素单体比例S/G均呈上升趋势,不同时期间达到极显著差异,说明S-型木质素占总木质素的比例越来越高；干旱胁迫下的茎秆木质素单体S/G的平均比例均略高于对照组,但各发育时期的差异均未达到显著水平；(6)初花期、终花期和青荚期两种环境条件下抗倒性材料的木质素单体S/G比率明显高于易倒伏材料,两种环境下,木质素单体S/G比率与茎杆抗折性间均达到了极显著正相关关系,推测S型木质素单体在木质素中所占的比例与油菜抗倒性有密切关系。
3)分析干旱胁迫下木质素代谢途径中关键基因表达特征发现。在油菜易发生倒伏的四个时期,每个材料在干旱组和对照组中各取3株,提取茎杆中部样品的RNA,采用荧光定量PCR法,测定不同处理下11个木质素代谢途径关键基因的相对表达量差异,结果表明：(1)在两种环境下,基因PAL1的表达量都是先升高后减小,但干旱胁迫下的表达量峰值推迟,在终花期明显高于正常环境；正常环境下,抗倒性较高材料在蕾薹期、终花期和青荚期基因PAL1表达量均明显高于易倒伏材料；(2)两种环境下,基因PAL2表达量都是先升高后降低,在初花期达到峰值,初花期正常环境下的PAL2表达量显著高于干旱胁迫环境,但终花期时的情况刚好相反；两种环境下,抗倒伏材料PAL2表达量在蕾薹期都明显高于易倒伏材料,其它时期差异不大；(3)两种环境下,基因C4H在茎秆中表达量从初花期到青荚期都是单调上升,正常环境略高于干旱胁迫；蕾薹期、终花期和青荚期易倒伏材料C4H的表达量明显高于抗倒伏材料,青荚期差异达到最大；(4)在两种环境下,基因4CL1的表达量变化趋势同PAL1;正常环境下,抗倒材料在蕾薹期和终花期4CL1的表达量明显高于易倒伏材料,胁迫条件下两类材料的差异不明显；(5)在两种环境下,基因4CL2表达量的变化趋势相同,在终花期达到峰值且干旱胁迫下的表达量明显高于正常环境；两类材料间的表达量差异同4CL1；(6)在两种环境下,基因COMT表达量变化趋势不同,干旱胁迫下为抛物线型,正常环境下为S型,在终花期干旱胁迫下的表达量显著高于正常环境；正常环境下,初花期和青荚期易倒伏材料的基因COMT表达量显著高于抗倒材料,但干旱胁迫下,青荚期易倒伏材料的基因COMT表达量却明显低于抗倒材料；(7)在两种环境下,基因CCoAOMT的表达量仅在青荚期迅速增加,且干旱胁迫下的表达量极显著高于正常环境；比较两类材料发现,两种环境下青荚期易倒伏材料基因CCoAOMT的表达量均远高于抗倒材料；(8)两种环境下基因F5H的表达量变化趋势相同,在初花期有峰值,但环境间差异不大；比较两类材料,抗倒材料在蕾薹期和终花期F5H的表达量明显高于易倒伏材料；(9)两种环境下基因CCRI表达量变化趋势与PAL1和4CL1表达特点相似；正常环境下,抗倒材料在蕾薹期和终花期的表达量明显高于易倒伏材料,干旱胁迫下,抗倒材料在终花期的表达量明显高于易倒伏材料；(10)干旱胁迫下,基因LAC4(?)的表达量在蕾苔期显著高于正常环境,但自初花期后,两种环境下的表达量都急剧下降,且两种环境下没有明显区别；两类材料间,仅在蕾苔期发现抗倒材料基因LAC4的表达量明显高于易倒伏材料；(11)两种环境下LAC17基因表达的变化特点完全同LAC4:两类材料比较发现,正常环境下,抗倒材料在终花期和青荚期的表达量明显高于易倒伏材料,但干旱胁迫下,抗倒材料在蕾苔期、终花期和青荚期的表达量却明显低于易倒伏材料。
4)基因表达量间及与木质素性状间的相关分析。分两种环境计算了11个基因表达量间以及与木质素含量、木质素单体S/G间的相关系数,并据此就11个基因作了聚类分析；以木质素含量、木质素单体S/G为目标性状,以11个基因表达量为自变量,分两种环境进行了通径分析。主要结果有：(1)正常环境下,各基因表达量间绝大多数有极显著正相关关系,表现明显的协同作用,只有COMT、 CCoAOMT基因较特殊,与大多数基因关系不明显,且与PAL2、LAC4和LAC17有负相关关系；在干旱胁迫下,C4H基因表达量只与CCoAOMT有极显著正相关,LAC4和LAC17只与PAL2、CCR1有极显著正相关,其余基因间保持明显的协同作用；(2)基因表达量聚类分析表明,LAC4和LAC17表达特征较一致,PAL1、4CL1.4CL2、CCR1和F5H的表达特点较一致,COMT、C4H和CCoAOMT的表达特征较特殊；(3)简单相关系数分析表明,两种环境下都只有基因LAC4和LAC17的表达量与木质素含量和木质素单体S/G间有极显著负相关,其余基因表达量与两个性状无明显关系；(4)通径分析表明,在正常环境下,基因PAL1的表达量与木质素含量作用的直接通径效应最大,其次是4CL2和4CL1；基因LAC17表达量与木质素的直接通径系数为负的最大值,其次是F5H和C4H。在干旱条件下,各基因的表达量与木质素的直接通径效应发生变化,基因CCoAOMT的表达量与木质素含量作用的直接通径效应最大,其次是4CL2和F5H；基因C4H的表达量与木质素的直接通径系数为负的最大值,其次是4CL1和LAC17。
上述研究结果初步明确了干旱胁迫对油菜茎秆水分含量、株高、直径、抗折力、木质素含量和木质素单体S/G在不同生育期产生的影响,初步分析了木质素合成各关键基因在不同生育期下表达量的动态变化规律,找到若干在干旱胁迫下的差异表达基因,为今后进一步阐明木质素代谢网络的环境调控、增加油菜茎杆木质素含量和抗折能力奠定了较好的基础。
The proportion of edible vegetable oil provided by ourselves is merely40%, which makes the rapeseed become one of the weakest bulk agricultural products in China. There are two predominant factors to restrict the increase of the rapeseed plant scale:the low resistance and low mechanization level. Lodging not only results in a decrease of rapeseed production which ranges from10%-30%and even more than50%, but also affect the quality that leads to a10%to30%decrease of the oil content. It is also an obstacle to mechanization level increasing. Drought is one of the climatic disasters for crop and agriculture production, the proportion of which in the distorted fields had been as high as75%.Thus, it is a very important approach to enhance the resistance of rape to drought and lodging by investigating the regulation and expression mechanism underlying lignin gene in rape stem under drought stress, which could also contribute to rape breeding for new variety that is wide tolerance, high resistance and more suitable for sowing and harvest by machine.
We have selected10species (lines) of Brassica napus with stable highest lodging resistance and lowest lodging resistance, to analyze the difference and dynamic changes of those morphology traits of stem, the lignin content and the rate of different lignin monomers in different development stage, by planting them in a shed and treated with drought condition and also set a control with plenty water. We compared the different expression of lignin metabolic pathway genes and revealed the mechanism for lodging resistance in drought condition.
The primary results are as follows:
1) Relationship between the traits related to lodging resistance and stem breaking-resistance under drought stress treatment. We collected3plants in each species to investigate the plant height, stem diameter, water content, breaking-resistance in four significant development stages of rapeseed (budding stage, early flowering stage, final flowering and podding stage). The dynamic changes for each trait in both treatments were analyzed, and the effects on breaking-resistance were also analyzed by path analysis. The results showed that:(1) there were significant difference in the performance of the four traits under drought stress and the control treatment. In drought stress environment the plants will be shorter, lower water content and smaller stem diameter, but with stronger breaking-resistance;(2) there also have obviously genotype-environment interaction effect those trails during some of the developmental stages;(3) in both environments plant height had largest direct effect on breaking-resistance, and stem diameter also has certain directly determine on breaking-resistance. The remaining path coefficients showed that here maybe other factors influence the stem breaking-resistance besides plant height and diameter.
2) The relationships of rapeseed stem breaking-resistance to the ratio of lignin monomers and lignin content in under drought stress. During four stages of the rapeseed development, we took3plants from the drought treatment and control treatment for each species, determined the ratio of lignin monomers and lignin content using the middle portion stem of the plant. The results show that:(1) The rapid accumulation period of the rapeseed stalk lignin was during the final flowering stage to podding stage;(2) Stem lignin content was greatly impacted by environment and different development stages. Stem lignin accumulation was accelerated under the drought conditions. There also obviously genotype-environment interaction effect to the accumulation of stem lignin except budding stage;(3)The correlation analysis of lignin content and breaking-resistance showed that there were highly significant positive correlation between the lignin content and stem breaking-resistance in both environmental conditions. It confirmed that the higher stem lignin content means the stronger mechanical strength, and the breaking-resistance;(4) The H-lignin accounted for10.96%of the total lignin components by average, and there was little changes in different species. The lignin monomers of rapeseed were mainly constituted by S-G lignin, the differences of the lignin monomer S/G ratio were very significant in the various species;(5) During the budding to the podding stage, S/G ratio of the lignin monomer showed an uptrend in two kinds of environments, and it shows highly significant difference between each stages, which showed that the S-lignin increasingly became the main monomer of the total lignin:The average S/G ratio of stem lignin monomer in the drought condition was slightly higher than control group, but the differences of S/G in various developmental stages haven't reached significant level. (6)In early flowering stage, final flowering stage and podding stage, the S/G ratio of lignin monomer in lodging resistance species was significantly higher than that of lower lodging-resistant species in both treatments. And the S/G ratio of lignin monomer showed a highly significantly positive correlation to the breaking-resistance of stem. It speculated that in rapeseed S-lignin monomer proportion may closely relate to the lodging resistance.
3) The expression analysis of key genes in the lignin metabolic pathways under drought stress. In the drought group and the control group, RNA of three plants stem of each species were extracted during four stages of rapeseed development. The expressions of11key genes in lignin metabolic pathways were analyzed by using qRT-PCR methods. The results showed that:(1) in both environments the expression of PALI gene will increase first and then decreased. The peak of expression under drought stress peak delayed under drought stress condition, and drought treatment can make PALI express significantly higher in the final flowering stage than normal environment. Under control treatment, the PALI expression of lodging-resistance species were significantly higher than that of lodging species in the budding stage, the final flowering and podding stage;(2) the expression of PAL2gene increased first and then decreased. There was a peak in the early flowering stage in both environments. In control condition the expression of PAL2was significantly higher than that in the drought stress environment, which was just opposite in the final flowering stage. In the budding stage the expression of PAL2of lodging-resistance species were significantly higher than lodging species, it doesn't shows significant difference in other stages;(3) the expression of C4H gene increasing from the early flowering stage to podding stage in both environments, and the expression in control condition always higher than the drought condition; the expression of lodging species was significantly higher than lodging-resistant species in budding stage, final flowering stage and podding stage, and in the podding stage the difference expression became maximized;(4) the expression characteristic of4CL1gene was similar with that of PALI in both environments. In controlled condition, the expression of lodging-resistance species was significantly higher than that of lodging species in the budding and final flowering stage, and there is no significant difference between two types of lodging-resistance species under drought stress conditions;(5) The expression of the gene4CL2trended to be identical in two environments, the maximum expression is in the final flowering stage, and the expression in drought stress condition obviously higher than that in the controlled condition; the expression characteristics between two types of lodging-resistance species were similar with gene4CL1;(6) the expression characteristics of the expression of gene COMT was different in two treatments, expression curve was like parabolic in drought stress condition, while S-type in controlled environment, in the final flowering stage there was significantly higher expression in drought stress condition than controlled condition, the expression of gene COMT in lodging species is obviously higher than that with lodging-resistant species in early flowering stage and podding stage, while in drought stress treatment the result was contrary in podding stage.(7) the expression of gene CCoAOMT rapidly increased in the podding stage in both treatments, and in drought stress the expression was significantly higher than in the controlled treatment. The expression of CCoAOMT gene in lodging species was significantly higher than that in lodging-resistance species.(8) The expression characteristic of F5H doesn't change in both environments, the expression maximum shows in the early flowering stage. The expression of lodging-resistance species was significant higher than the lodging species in the budding and final flowering stage;(9) the expression characteristic of gene CCR1was similar with that of PAL1and4CL1in both environments. In controlled treatment, the expression of lodging-resistance species was significantly higher than that of lodging species in the budding and final flowering stage. In final flowering stage the expression of lodging-resistance species was significantly higher than lodging species under drought stress;(10) in the budding stage the expression of the gene LAC4under drought stress was significantly higher than that in the controlled condition, but after the early flowering stage the expression decreased sharply in both environments, and no significant difference between them:only in the budding stage the expression of lodging-resistance species was significantly higher than that of lodging species;(11) the expression characteristic of LAC17was similar with LAC4expression under two conditions. The expression of lodging-resistance species was significantly higher than lodging plants in final flowering and podding stage under normal circumstances. But, under drought stress, the expression of lodging-resistance species was much lower than lodging species in budding, final flowering and podding stages.
4) Correlation analysis gene expression and lignin traits.We had correlation analysis11genes expression with each other and those genes expression with lignin content and lignin monomer S/G, In addition, a cluster analysis of these11genes expression character was build. We use the path analysis to investigated target traits of lignin content and lignin monomer S/G with11genes expression as independent variables in each treatment condition. The main results are as follows:(1) except COMT and CCoAOMT, there were significant positive correlation and apparent synergy between most genes. COMT, CCoAOMT was no obvious correlation with other genes; even have negative correlation with PAL2and LAC4expression characters; In the drought stress treatment, there were significant positive correlations between C4H and CCoAOMT, LAC4and LAC17only significant positive correlation with PAL1and PAL2, in other genes still shows the apparent synergy.(2) Gene expression cluster analysis showed that:LAC4and LAC17expression characteristics were similar and PALI,4CL1,4CL2, CCR1and F5H genes expression were similar to each other, but COMT, C4H and CCoAOMT had their own expression characteristics.(3) The correlation coefficient analysis showed that there were significant negative correlations between LAC4and LAC17to the lignin content and lignin monomer S/G, but others genes have no obvious relationship with them;(4) The results of path analysis indicate that:PALI contribute the most direct effect to lignin content, next were4CL2and4CL1;Contrarily, LAC17expression contributes the largest negative effect to lignin content, and the next were F5H and C4H. The direct effect of these genes expression to the lignin content had changed under drought stress. CCoAOMT contribute the most direct effect to lignin content, next were4CL2and F5H; C4H expression contributes the largest negative effect to lignin content, and next were4CL1and LAC17.
We clarified the effects on water content, plant height, diameter, breaking-resistant strength, lignin content and the S/G in drought stress condition; analyzed the dynamic changes of the of lignin metabolic pathways genes expression in different development stage; identified the genes with different expression genes between two conditions. All these results will be benefit for a further understanding on the regulation of lignin metabolic, the network of lignin related genes, and to increasing lignin content in stem for the enhance of lodging resistance in rapeseed.
本研究以10份较稳定的抗倒伏和易倒伏极端表现型甘蓝型油菜品种(系)为材料,通过旱棚内干旱胁迫和正常水分对照的对比试验,分析了抗倒伏与易倒伏极端表现型材料的茎秆相关性状、茎杆木质素含量及木质素单体比例等性状在不同发育时期的动态变化规律,比较了不同发育时期茎秆中木质素合成相关基因的表达差异,初步揭示了干旱胁迫下甘蓝型油菜抗倒伏特性形成的机理。主要研究结果如下：
1)干旱胁迫下油菜抗倒伏相关性状与茎杆抗折力间的关系。在油菜易发生倒伏的四个时期(蕾苔期、初花期、终花期、青荚期),每个材料在干旱组和对照组中各取3株,调查了株高、茎杆中部直径、含水量、抗折断力等性状,详细分析了各性状在两种环境下的动态变化差异；采用通径分析方法分析了株高、茎杆中部直径、含水量对茎杆抗折断力的影响。结果发现：(1)四个性状在干旱胁迫下的表现均与正常环境有明显差别,一般趋势是,干旱胁迫环境下,油菜植株变矮、茎杆含水量减少、茎杆直径变小但抗折断力增加；(2)各参试材料除受环境影响有共同的变化趋势以外,在部分发育时期有明显的基因型-环境互作现象存在；(3)通径分析表明在两种环境下,油菜的株高对茎秆的抗折力有最大的直接作用,直径对茎秆的抗折力也有一定的直接决定作用,剩余通径系数表明除株高和直径外还有其他一些因素决定茎秆的抗折能力。
2)干旱胁迫下油菜茎杆木质素含量、单体比率与油菜茎杆抗折性关系。在油菜易发生倒伏的四个时期,每个材料在于旱组和对照组中各取3株,测定了中部茎秆的木质素含量和木质素单体比例。结果表明：(1)终花期至青荚期间是油菜茎秆木质素的快速积累期；(2)在不同时期和不同环境处理下,茎杆木质素含量受环境影响很大,除蕾苔期外茎杆木质素在干旱条件下合成积累加快,其余时期茎杆木质素的合成速率还与基因型有关,存在明显的基因型-环境互作现象；(3)木质素含量与抗折性间的相关分析表明,两种环境条件下,油菜茎秆木质素的含量与茎秆抗折性间均存在显著或极显著的正相关关系,说明茎杆木质素含量愈高,茎秆的机械强度愈强,抗折性也愈强；(4)油菜H型木质素平均仅占总木质素总成分的10.96%,且各材料间相差不大；油菜木质素单体主要为S-G型木质素,各材料间木质素单体S/G比率有极显著差异；(5)蕾薹期至青荚期,两种环境下参试材料的主要木质素单体比例S/G均呈上升趋势,不同时期间达到极显著差异,说明S-型木质素占总木质素的比例越来越高；干旱胁迫下的茎秆木质素单体S/G的平均比例均略高于对照组,但各发育时期的差异均未达到显著水平；(6)初花期、终花期和青荚期两种环境条件下抗倒性材料的木质素单体S/G比率明显高于易倒伏材料,两种环境下,木质素单体S/G比率与茎杆抗折性间均达到了极显著正相关关系,推测S型木质素单体在木质素中所占的比例与油菜抗倒性有密切关系。
3)分析干旱胁迫下木质素代谢途径中关键基因表达特征发现。在油菜易发生倒伏的四个时期,每个材料在干旱组和对照组中各取3株,提取茎杆中部样品的RNA,采用荧光定量PCR法,测定不同处理下11个木质素代谢途径关键基因的相对表达量差异,结果表明：(1)在两种环境下,基因PAL1的表达量都是先升高后减小,但干旱胁迫下的表达量峰值推迟,在终花期明显高于正常环境；正常环境下,抗倒性较高材料在蕾薹期、终花期和青荚期基因PAL1表达量均明显高于易倒伏材料；(2)两种环境下,基因PAL2表达量都是先升高后降低,在初花期达到峰值,初花期正常环境下的PAL2表达量显著高于干旱胁迫环境,但终花期时的情况刚好相反；两种环境下,抗倒伏材料PAL2表达量在蕾薹期都明显高于易倒伏材料,其它时期差异不大；(3)两种环境下,基因C4H在茎秆中表达量从初花期到青荚期都是单调上升,正常环境略高于干旱胁迫；蕾薹期、终花期和青荚期易倒伏材料C4H的表达量明显高于抗倒伏材料,青荚期差异达到最大；(4)在两种环境下,基因4CL1的表达量变化趋势同PAL1;正常环境下,抗倒材料在蕾薹期和终花期4CL1的表达量明显高于易倒伏材料,胁迫条件下两类材料的差异不明显；(5)在两种环境下,基因4CL2表达量的变化趋势相同,在终花期达到峰值且干旱胁迫下的表达量明显高于正常环境；两类材料间的表达量差异同4CL1；(6)在两种环境下,基因COMT表达量变化趋势不同,干旱胁迫下为抛物线型,正常环境下为S型,在终花期干旱胁迫下的表达量显著高于正常环境；正常环境下,初花期和青荚期易倒伏材料的基因COMT表达量显著高于抗倒材料,但干旱胁迫下,青荚期易倒伏材料的基因COMT表达量却明显低于抗倒材料；(7)在两种环境下,基因CCoAOMT的表达量仅在青荚期迅速增加,且干旱胁迫下的表达量极显著高于正常环境；比较两类材料发现,两种环境下青荚期易倒伏材料基因CCoAOMT的表达量均远高于抗倒材料；(8)两种环境下基因F5H的表达量变化趋势相同,在初花期有峰值,但环境间差异不大；比较两类材料,抗倒材料在蕾薹期和终花期F5H的表达量明显高于易倒伏材料；(9)两种环境下基因CCRI表达量变化趋势与PAL1和4CL1表达特点相似；正常环境下,抗倒材料在蕾薹期和终花期的表达量明显高于易倒伏材料,干旱胁迫下,抗倒材料在终花期的表达量明显高于易倒伏材料；(10)干旱胁迫下,基因LAC4(?)的表达量在蕾苔期显著高于正常环境,但自初花期后,两种环境下的表达量都急剧下降,且两种环境下没有明显区别；两类材料间,仅在蕾苔期发现抗倒材料基因LAC4的表达量明显高于易倒伏材料；(11)两种环境下LAC17基因表达的变化特点完全同LAC4:两类材料比较发现,正常环境下,抗倒材料在终花期和青荚期的表达量明显高于易倒伏材料,但干旱胁迫下,抗倒材料在蕾苔期、终花期和青荚期的表达量却明显低于易倒伏材料。
4)基因表达量间及与木质素性状间的相关分析。分两种环境计算了11个基因表达量间以及与木质素含量、木质素单体S/G间的相关系数,并据此就11个基因作了聚类分析；以木质素含量、木质素单体S/G为目标性状,以11个基因表达量为自变量,分两种环境进行了通径分析。主要结果有：(1)正常环境下,各基因表达量间绝大多数有极显著正相关关系,表现明显的协同作用,只有COMT、 CCoAOMT基因较特殊,与大多数基因关系不明显,且与PAL2、LAC4和LAC17有负相关关系；在干旱胁迫下,C4H基因表达量只与CCoAOMT有极显著正相关,LAC4和LAC17只与PAL2、CCR1有极显著正相关,其余基因间保持明显的协同作用；(2)基因表达量聚类分析表明,LAC4和LAC17表达特征较一致,PAL1、4CL1.4CL2、CCR1和F5H的表达特点较一致,COMT、C4H和CCoAOMT的表达特征较特殊；(3)简单相关系数分析表明,两种环境下都只有基因LAC4和LAC17的表达量与木质素含量和木质素单体S/G间有极显著负相关,其余基因表达量与两个性状无明显关系；(4)通径分析表明,在正常环境下,基因PAL1的表达量与木质素含量作用的直接通径效应最大,其次是4CL2和4CL1；基因LAC17表达量与木质素的直接通径系数为负的最大值,其次是F5H和C4H。在干旱条件下,各基因的表达量与木质素的直接通径效应发生变化,基因CCoAOMT的表达量与木质素含量作用的直接通径效应最大,其次是4CL2和F5H；基因C4H的表达量与木质素的直接通径系数为负的最大值,其次是4CL1和LAC17。
上述研究结果初步明确了干旱胁迫对油菜茎秆水分含量、株高、直径、抗折力、木质素含量和木质素单体S/G在不同生育期产生的影响,初步分析了木质素合成各关键基因在不同生育期下表达量的动态变化规律,找到若干在干旱胁迫下的差异表达基因,为今后进一步阐明木质素代谢网络的环境调控、增加油菜茎杆木质素含量和抗折能力奠定了较好的基础。
The proportion of edible vegetable oil provided by ourselves is merely40%, which makes the rapeseed become one of the weakest bulk agricultural products in China. There are two predominant factors to restrict the increase of the rapeseed plant scale:the low resistance and low mechanization level. Lodging not only results in a decrease of rapeseed production which ranges from10%-30%and even more than50%, but also affect the quality that leads to a10%to30%decrease of the oil content. It is also an obstacle to mechanization level increasing. Drought is one of the climatic disasters for crop and agriculture production, the proportion of which in the distorted fields had been as high as75%.Thus, it is a very important approach to enhance the resistance of rape to drought and lodging by investigating the regulation and expression mechanism underlying lignin gene in rape stem under drought stress, which could also contribute to rape breeding for new variety that is wide tolerance, high resistance and more suitable for sowing and harvest by machine.
We have selected10species (lines) of Brassica napus with stable highest lodging resistance and lowest lodging resistance, to analyze the difference and dynamic changes of those morphology traits of stem, the lignin content and the rate of different lignin monomers in different development stage, by planting them in a shed and treated with drought condition and also set a control with plenty water. We compared the different expression of lignin metabolic pathway genes and revealed the mechanism for lodging resistance in drought condition.
The primary results are as follows:
1) Relationship between the traits related to lodging resistance and stem breaking-resistance under drought stress treatment. We collected3plants in each species to investigate the plant height, stem diameter, water content, breaking-resistance in four significant development stages of rapeseed (budding stage, early flowering stage, final flowering and podding stage). The dynamic changes for each trait in both treatments were analyzed, and the effects on breaking-resistance were also analyzed by path analysis. The results showed that:(1) there were significant difference in the performance of the four traits under drought stress and the control treatment. In drought stress environment the plants will be shorter, lower water content and smaller stem diameter, but with stronger breaking-resistance;(2) there also have obviously genotype-environment interaction effect those trails during some of the developmental stages;(3) in both environments plant height had largest direct effect on breaking-resistance, and stem diameter also has certain directly determine on breaking-resistance. The remaining path coefficients showed that here maybe other factors influence the stem breaking-resistance besides plant height and diameter.
2) The relationships of rapeseed stem breaking-resistance to the ratio of lignin monomers and lignin content in under drought stress. During four stages of the rapeseed development, we took3plants from the drought treatment and control treatment for each species, determined the ratio of lignin monomers and lignin content using the middle portion stem of the plant. The results show that:(1) The rapid accumulation period of the rapeseed stalk lignin was during the final flowering stage to podding stage;(2) Stem lignin content was greatly impacted by environment and different development stages. Stem lignin accumulation was accelerated under the drought conditions. There also obviously genotype-environment interaction effect to the accumulation of stem lignin except budding stage;(3)The correlation analysis of lignin content and breaking-resistance showed that there were highly significant positive correlation between the lignin content and stem breaking-resistance in both environmental conditions. It confirmed that the higher stem lignin content means the stronger mechanical strength, and the breaking-resistance;(4) The H-lignin accounted for10.96%of the total lignin components by average, and there was little changes in different species. The lignin monomers of rapeseed were mainly constituted by S-G lignin, the differences of the lignin monomer S/G ratio were very significant in the various species;(5) During the budding to the podding stage, S/G ratio of the lignin monomer showed an uptrend in two kinds of environments, and it shows highly significant difference between each stages, which showed that the S-lignin increasingly became the main monomer of the total lignin:The average S/G ratio of stem lignin monomer in the drought condition was slightly higher than control group, but the differences of S/G in various developmental stages haven't reached significant level. (6)In early flowering stage, final flowering stage and podding stage, the S/G ratio of lignin monomer in lodging resistance species was significantly higher than that of lower lodging-resistant species in both treatments. And the S/G ratio of lignin monomer showed a highly significantly positive correlation to the breaking-resistance of stem. It speculated that in rapeseed S-lignin monomer proportion may closely relate to the lodging resistance.
3) The expression analysis of key genes in the lignin metabolic pathways under drought stress. In the drought group and the control group, RNA of three plants stem of each species were extracted during four stages of rapeseed development. The expressions of11key genes in lignin metabolic pathways were analyzed by using qRT-PCR methods. The results showed that:(1) in both environments the expression of PALI gene will increase first and then decreased. The peak of expression under drought stress peak delayed under drought stress condition, and drought treatment can make PALI express significantly higher in the final flowering stage than normal environment. Under control treatment, the PALI expression of lodging-resistance species were significantly higher than that of lodging species in the budding stage, the final flowering and podding stage;(2) the expression of PAL2gene increased first and then decreased. There was a peak in the early flowering stage in both environments. In control condition the expression of PAL2was significantly higher than that in the drought stress environment, which was just opposite in the final flowering stage. In the budding stage the expression of PAL2of lodging-resistance species were significantly higher than lodging species, it doesn't shows significant difference in other stages;(3) the expression of C4H gene increasing from the early flowering stage to podding stage in both environments, and the expression in control condition always higher than the drought condition; the expression of lodging species was significantly higher than lodging-resistant species in budding stage, final flowering stage and podding stage, and in the podding stage the difference expression became maximized;(4) the expression characteristic of4CL1gene was similar with that of PALI in both environments. In controlled condition, the expression of lodging-resistance species was significantly higher than that of lodging species in the budding and final flowering stage, and there is no significant difference between two types of lodging-resistance species under drought stress conditions;(5) The expression of the gene4CL2trended to be identical in two environments, the maximum expression is in the final flowering stage, and the expression in drought stress condition obviously higher than that in the controlled condition; the expression characteristics between two types of lodging-resistance species were similar with gene4CL1;(6) the expression characteristics of the expression of gene COMT was different in two treatments, expression curve was like parabolic in drought stress condition, while S-type in controlled environment, in the final flowering stage there was significantly higher expression in drought stress condition than controlled condition, the expression of gene COMT in lodging species is obviously higher than that with lodging-resistant species in early flowering stage and podding stage, while in drought stress treatment the result was contrary in podding stage.(7) the expression of gene CCoAOMT rapidly increased in the podding stage in both treatments, and in drought stress the expression was significantly higher than in the controlled treatment. The expression of CCoAOMT gene in lodging species was significantly higher than that in lodging-resistance species.(8) The expression characteristic of F5H doesn't change in both environments, the expression maximum shows in the early flowering stage. The expression of lodging-resistance species was significant higher than the lodging species in the budding and final flowering stage;(9) the expression characteristic of gene CCR1was similar with that of PAL1and4CL1in both environments. In controlled treatment, the expression of lodging-resistance species was significantly higher than that of lodging species in the budding and final flowering stage. In final flowering stage the expression of lodging-resistance species was significantly higher than lodging species under drought stress;(10) in the budding stage the expression of the gene LAC4under drought stress was significantly higher than that in the controlled condition, but after the early flowering stage the expression decreased sharply in both environments, and no significant difference between them:only in the budding stage the expression of lodging-resistance species was significantly higher than that of lodging species;(11) the expression characteristic of LAC17was similar with LAC4expression under two conditions. The expression of lodging-resistance species was significantly higher than lodging plants in final flowering and podding stage under normal circumstances. But, under drought stress, the expression of lodging-resistance species was much lower than lodging species in budding, final flowering and podding stages.
4) Correlation analysis gene expression and lignin traits.We had correlation analysis11genes expression with each other and those genes expression with lignin content and lignin monomer S/G, In addition, a cluster analysis of these11genes expression character was build. We use the path analysis to investigated target traits of lignin content and lignin monomer S/G with11genes expression as independent variables in each treatment condition. The main results are as follows:(1) except COMT and CCoAOMT, there were significant positive correlation and apparent synergy between most genes. COMT, CCoAOMT was no obvious correlation with other genes; even have negative correlation with PAL2and LAC4expression characters; In the drought stress treatment, there were significant positive correlations between C4H and CCoAOMT, LAC4and LAC17only significant positive correlation with PAL1and PAL2, in other genes still shows the apparent synergy.(2) Gene expression cluster analysis showed that:LAC4and LAC17expression characteristics were similar and PALI,4CL1,4CL2, CCR1and F5H genes expression were similar to each other, but COMT, C4H and CCoAOMT had their own expression characteristics.(3) The correlation coefficient analysis showed that there were significant negative correlations between LAC4and LAC17to the lignin content and lignin monomer S/G, but others genes have no obvious relationship with them;(4) The results of path analysis indicate that:PALI contribute the most direct effect to lignin content, next were4CL2and4CL1;Contrarily, LAC17expression contributes the largest negative effect to lignin content, and the next were F5H and C4H. The direct effect of these genes expression to the lignin content had changed under drought stress. CCoAOMT contribute the most direct effect to lignin content, next were4CL2and F5H; C4H expression contributes the largest negative effect to lignin content, and next were4CL1and LAC17.
We clarified the effects on water content, plant height, diameter, breaking-resistant strength, lignin content and the S/G in drought stress condition; analyzed the dynamic changes of the of lignin metabolic pathways genes expression in different development stage; identified the genes with different expression genes between two conditions. All these results will be benefit for a further understanding on the regulation of lignin metabolic, the network of lignin related genes, and to increasing lignin content in stem for the enhance of lodging resistance in rapeseed.
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赵华燕,魏建华,张景昱,刘惠荣,王台,宋艳茹.抑制COMT和CCoAOMT调控植物木质素的生物合成.科学通报,2002,47(8)：604-607
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Chapple CCS, Vogt T, Ellis BE, Somerville CR. An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell,1992,4:1413-1424
Chen AH (陈安和),Chai YR(柴友荣),Li JN(李加纳),Chen L(谌利).Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus), J Biochem Mol Biol,2007,40(2):247-260
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赵春桥.能源植物木质素组成结构对降解转化效率影响的研究[硕士学位论文].华中农业大学,2011.06
赵华燕,魏建华,张景昱,刘惠荣,王台,宋艳茹.抑制COMT和CCoAOMT调控植物木质素的生物合成.科学通报,2002,47(8)：604-607
张文华,甘蓝型油菜抗倒伏相关性状的遗传分析和QTL定位.[硕士学位论文].2010.06
Abbott JC, Barakate A, Pincon G, Legrand M, Lapierre C, Mila I. Schuch W, Halpin C. Simultaneous suppression of multiple genes by single transgenes down-regulation of three unrelated lignin biosynthetic genes in tobacco. Plant Physiol,2002,128:844-853
Anterola A M, Lewis N G. Trends in lignin modification:a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity [J]. Phytochemistry, 2002b,61:221-294.
Bate NJ, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner PW, Dixon RA, Lamb CJ, Elkind Y. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proc Natl Acad Sci U S A,1994,91(16):7608-7612
Baucher M, Monties B, Van M M, Boerjan W. Biosynthesis and genetic engineer in lignin. Critical Rev Plant Sci,1998,17:125-197
Baucher M, Christensen J H, Meyermans H, Chen C Y, Van Doorsselaere J, J C Leple, G Pilate, Petit-Conil M, Jouanin L, Chabbert B, Monties B, Van Montagu M, Boerjan W. Applications of molecular genetics for biosynthesis of novel lignins [J]. Polym Degrad Stabil,1998a,59:47-52.
Baucher M, Monties B, Van Montagu M, Boerjan W. Biosynthesis and genetic engineering of lignin [J]. Crit Rev Plant Sci,1998b,17:125-197.
Bhinu VS, Li R, Huang J, Kaminskyj S, Sharpe A, Hannoufa A. Perturbation of lignin biosynthesis pathway in Brassica napus (canola) plants using RNAi. Can J Plant Sci,2009,89:441-453
Bugos R, Chiang V, Campbell W. cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen. Plant Mol Biol,1991,17:1203-1215
Chabannes M, Barakate A, Lapierre C, Marita JM, Ralph J, Pean M, Danoun S, Halpin C, Grima-Pettenati J, Boudet AM. Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants. Plant J,2001,28: 257-270
Chapple CCS, Vogt T, Ellis BE, Somerville CR. An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell,1992,4:1413-1424
Chen AH (陈安和),Chai YR(柴友荣),Li JN(李加纳),Chen L(谌利).Molecular cloning of two genes encoding cinnamate 4-hydroxylase (C4H) from oilseed rape (Brassica napus), J Biochem Mol Biol,2007,40(2):247-260
Fan L, Linker R, Gepstein S, Tanimoto E, Yamamoto R, Neumann P M. Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics [J]. Plant Physiol,2006,140:603-612.
Franke R, McMichael C M, Meyer K, Shirley AM, Cusumano JC, Chapple C. Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase. Plant J,2000,22(3):223-234
Freudenberg K, Neish AC (eds). Constitution and Biosynthesis of Lignin. Springer-Verlag, New York,1968
Fu FY(付福友),Liu LZ(刘列钊),Chai YR (柴友荣),Chen L (湛利),Yang T(杨涛),Ma AF(马爱芬),Qu CM(曲存民)Jiang L(蒋林),Zhang ZS(张正圣), Li JN(李加纳)Localization of QTLs for husk proportion and lignin content using a high-density genetic linkage map of Brassica napus. Korean J Gene,2007,29(3):343-353.
Gang D R, Fujita M, Davin L B. Lewis NG. The "abnormal lignins":mapping heartwood formation through the lignan biosynthetic pathway [A]. In:Lewis. N.G., Sarkanen. S. (Eds.), Lignin and Lignan Biosynthesis, ACS Symposium Series Washington, DC,1998,697:389-421.
Goujon T. Ferret V, Mila I. Pollet B, Ruel K, Burlat V. Joseleau J P, Barriere Y. Lapierre C, Jouanin L. Down-regulation of the AtCCRl gene in Arabidopsis thaliana:effects on phenotype, lignins and cell wall degradability [J]. Planta,2003a.217:218-228.
Goujon T, Sibout R. Eudes A. MacKay J, Jouanin L. Genes involved in the biosynthesis of lignin precursors in Arabidopsis thaliana [J]. Plant Physiol Biochem.2003b.41:677-687.
Guo D, Chen F, Inoue K, Blount J, Dixon R. Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa:impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell,2001,13(1):73-88
Hu WJ, Harding SA. Lung J, Popko JL, Ralph J, Stokke DD, Tsai CJ, Chiang VL. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotech,1999,17(8):808-812
Humphreys JM. Chapple C. Rewriting the lignin roadmap. Curr Opin Plant Biol,2002.5(3): 224-229
Halpin C, Knight M E, Foxon G A. Manipulation of lignin quality by down-regulation of cinnamyl alcohol dehydrogenase. Plant J.1994,6:339-350
Henderson S J. Friend J. Increases in phenylalanine ammonia-Iyase activity and lignin-like compounds as race-specific-resistance responses of potato tubers to Phytophthora infestans [J]. Biochem Soc Trans,1978,6:393
Hu W J, Harding S A, Lung J, Popko J L. Ralph J, Stokke D D, Tsai C J, Chiang V L. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees [J]. Nat Biotechnol,1999,17:808-812.
Humphreys, J M, Hemm M R, Chapple C. New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase [J]. Proc Natl Acad Sci USA,1999,96: 10045-10050.
Jiang W-M(姜维梅), Zhang D-Q(张冬青),Xu C-X(徐春霄).Studies on the stem anatomy of Brsssica oil to lodging. J Zhejiang Agric Univ (Agric Life Sci),2001,27(4):439-442
Jones L, Ennos AR, Turner SR. Cloning and characterization of irregular xylem4 (irx4):a severely lignin-deficient mutant of Arabidopsis. Plant J,2001,26(2):205-216
Kajita S, Hishiyama S, Tomimura Y, Katayama Y, Omori S. Structural characterization of modified lignin in transgenic tobacco plants in which the activity of 4-coumarate:coenzyme A ligase is depressed [J]. Plant Physiol 1997a,114:871-879.
Kajita S, Katayama Y, Omori S. Alterations in the biosynthesis of lignin in transgenic plants with chimeric genes for 4-coumarate:coenzyme A ligase [J]. Plant Cell Physiol,1996,37:957-965.
Kajita S, Mashino Y, Nishikubo N, Katayama Y, Omori S. Imunological characterization of transgenic tobacco plants with a chimeric gene for 4-coumarate:CoA ligase that have altered lignin in their xylem tissue [J]. Plant Sci,1997b,128:109-118.
Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Wakabayashi K, Takahashi H, Umemura K, Umezawa T, Shimamoto K. Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice [J]. Proc Natl Acad Sci USA,2006, 1:230-235.
Lacombe E, Hawkins S, Van Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, Boudet A M, Grima-Pettenati J. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway:cloning, expression and phylogenetic relationships [J]. Plant J,1997,11: 429-441.
Lange B M, Lapierre C, Sandermann H Jr. Elicitor-induced spruce stress lignin (structural similarity to early developmental lignins) [J]. Plant Physiol,1995,108:1277-1287.
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