歪头菜对UV-B辐射增强的响应及其cDNA文库构建
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摘要
青藏高原地区多年来长期维持在一个臭氧低值状态,紫外线辐射能量积累很高,这对生活在这一地区的动植物会产生深刻影响。为研究增强的UV-B(280-320nm)辐射对该地区物种生长繁殖的影响,我们以野生豆科牧草歪头菜为研究材料,在实验室条件下研究了其在6.4KJ.m-2.d-1的UV-B辐射剂量下(模拟甘南地区夏季晴朗天空9%的臭氧层衰减)生长发育、生理生化、基因表达三个方面的变化情况。处理期间测量了植物的株高、地上生物量、叶面积、内源激素、植物养分、木质素合成相关基因表达量等指标。结果显示：
1)UV-B辐射增强处理使歪头菜叶片出现叶形歪斜、不对称现象,叶片表面出现褐色斑点,且茎部木质素层沉积加厚,茎中央髓腔增大,老化加重。
2)短时间的UV-B处理(10天)对歪头菜株高和地上生物量无明显影响,随着紫外处理时间的延长和辐射量的累积,株高和地上生物量开始显著低于对照组。UV-B辐射处理初期对歪头菜根的生长有较强抑制作用,在处理后期,歪头菜植株主要以根的生长为主,地上部分的生长变化较小。UV-B辐射显著降低了歪头菜叶面积。
3)植物内源激素ZT、6-BA、GAs、IAA在UV-B处理10天左右时,含量均显著高于对照组。10天以后,随着辐照时间的延长而迅速下降,并且显著低于对照组,其中GAs和IAA含量变化最为剧烈。UV-B处理使ABA含量显著升高。
4)全氮含量虽然在紫外处理的前10天内高于对照组,但随着紫外处理时间的延长,氮含量下降的速度快于对照组,说明紫外辐照加快了植物氮的消耗。有机碳含量在UV-B辐照处理的前10天内显著高于对照组,此后含量与对照组无明显区别。全磷含量变化同有机碳含量变化基本一致,表明歪头菜有机碳和全氮含量受UV-B辐照增强处理的影响较小。
5)CAD基因在UV-B辐射处理的整个过程中表达量均低于对照组,表达被抑制,对UV-B辐射反应较为敏感。F5H基因在UV-B处理的整个过程中表达水平下调。CCOAOMT基因表达量在紫外处理的整个过程中呈现上升趋势,这表明该基因易受到UV-B辐射激发,是调控歪头菜木质素合成较关键的基因。
同时,用SMART技术构建了歪头菜cDNA文库。经测定,初级文库滴度达到2.3×106pfu.mL-1,扩增后文库总滴度为6.2×109pfu.mL-1,重组率为92.7%。从扩增后的文库随机挑取16个噬菌斑进行PCR扩增鉴定,结果显示,插入片段大多分布在500～2500bp之间。用PCR法从该文库中扩增出了本实验室以往用RACE法克隆出的歪头菜木质素合成相关基因CAD3′端全序列。通过各项指标验证成功构建了歪头菜叶片全长cDNA文库。
A decrease in the ozone layer could lead to a significant increase in ultraviolet-B (UV-B) radiation (280-320nm) reaching the earth's surface. Recently an "ozone hole" has been discovered above the Qinghai-Tibet Plateau inChina. In order to examine the effects of the enhanced UV-B radiation on the growth and reproduction of the alpine species on Qinghai-Tibet Pleteau, we use wild legume forages-Vicia unijuga A as research materials in laboratory conditions with the UV-B radiation level was6.4K.J.m-2.d-1(simulating a9%ozone depletion over Gannan,China34。55'N,2900m). The growth and development, physiology and biochemistry, gene expression under UV-B radiation were explored in this study. The plant height, aboveground biomass, leaf area, endogenous hormones, plant nutrients, lignin synthesis-related gene expression levels and other indicators were measured. The results showed:
1) Enhanced UV-B radiation made the leafes of Vicia unijuga A skew, asymmetries, brown spots on the surface, and lignin deposition thickened in stems. In the central of stem, the medullary cavity was increase.
2) A short time of enhanced UV-B treatment (10days) had no significant effect in plant height and aboveground biomass of Vicia unijugaA, with the accumulation of UV-B treatment time and the amount of radiation, they was lower than that of the control group significantly. Early UV-B radiation treatment has a stronger inhibitory effect on the growth of root of Vicia unijuga A, in the later period, it was the root growth frist. And UV-B radiation reduced the leaves area of Vicia unijuga A significantly.
3) In10days of UV-B treatment, the plant endogenous hormones ZT,6-BA, GAs, IAA were higher than the control group significantly.10days later, the content of them were decreased rapidly and significantly lower than the control group with the irradiation time. Changes in the content of GAs and IAA were most dramatic. UV-B treatment increased the ABA content significantly.
4) Total nitrogen content was higher than that of the control group in the first10days, but with the time extension of UV-B treatment, the rate of decline of total nitrogen is faster than the control group, which indicated that UV-B irradiation accelerated the consumption of plant total nitrogen. The content of organic carbon was higher than the control group significantly in the first10days of UV-B irradiation treatment, after which, the content was no significant difference between the control group. The content of total phosphorus is consistent with the changes in the content of organic carbon, which showed that the content of organic carbon and nitrogen of Vicia unijuga A have a less affected by UV-B irradiation enhanced.
5) In the whole process of UV-B radiation, the express of CAD gene was lower than the control group, the expression is suppressed, it's more sensitive to UV-B radiation. Despite the express of F5H gene has an increase in the whole process of the UV-B, compared to the expression level of the control group, it's still suppressed. Gene expression of CCOAOMT in the whole process of UV-B radiation presents a rising trend, which shows that the gene is susceptible to UV-B radiation excited, and it is the key gene in lignin synthesis.
At the same time, we constructed a full-length cDNA Library of Vicia unijuga A by SMART technique. According to the evaluation on quality, The titer of primary library was2.3×106pfu·mL-1, while the amplified library has a high titer of6.2×109pfu·mL-1and92.7%recombinant percentage.16plaques were randomly picked from the amplified library, after amplified by PCR identification, the results showed that the inserted fragment is mostly distributed between500to2500bp. The3'end full-length cDNA of CAD, a Vicia unijuga A gene related with lignin synthesis cloned by RACE method in the past, was amplified in this cDNA Library by PCR, which indicated that the cDNA library for leaves of Vicia unijuga had been constructed successfully by using the SMART technique.
1)UV-B辐射增强处理使歪头菜叶片出现叶形歪斜、不对称现象,叶片表面出现褐色斑点,且茎部木质素层沉积加厚,茎中央髓腔增大,老化加重。
2)短时间的UV-B处理(10天)对歪头菜株高和地上生物量无明显影响,随着紫外处理时间的延长和辐射量的累积,株高和地上生物量开始显著低于对照组。UV-B辐射处理初期对歪头菜根的生长有较强抑制作用,在处理后期,歪头菜植株主要以根的生长为主,地上部分的生长变化较小。UV-B辐射显著降低了歪头菜叶面积。
3)植物内源激素ZT、6-BA、GAs、IAA在UV-B处理10天左右时,含量均显著高于对照组。10天以后,随着辐照时间的延长而迅速下降,并且显著低于对照组,其中GAs和IAA含量变化最为剧烈。UV-B处理使ABA含量显著升高。
4)全氮含量虽然在紫外处理的前10天内高于对照组,但随着紫外处理时间的延长,氮含量下降的速度快于对照组,说明紫外辐照加快了植物氮的消耗。有机碳含量在UV-B辐照处理的前10天内显著高于对照组,此后含量与对照组无明显区别。全磷含量变化同有机碳含量变化基本一致,表明歪头菜有机碳和全氮含量受UV-B辐照增强处理的影响较小。
5)CAD基因在UV-B辐射处理的整个过程中表达量均低于对照组,表达被抑制,对UV-B辐射反应较为敏感。F5H基因在UV-B处理的整个过程中表达水平下调。CCOAOMT基因表达量在紫外处理的整个过程中呈现上升趋势,这表明该基因易受到UV-B辐射激发,是调控歪头菜木质素合成较关键的基因。
同时,用SMART技术构建了歪头菜cDNA文库。经测定,初级文库滴度达到2.3×106pfu.mL-1,扩增后文库总滴度为6.2×109pfu.mL-1,重组率为92.7%。从扩增后的文库随机挑取16个噬菌斑进行PCR扩增鉴定,结果显示,插入片段大多分布在500～2500bp之间。用PCR法从该文库中扩增出了本实验室以往用RACE法克隆出的歪头菜木质素合成相关基因CAD3′端全序列。通过各项指标验证成功构建了歪头菜叶片全长cDNA文库。
A decrease in the ozone layer could lead to a significant increase in ultraviolet-B (UV-B) radiation (280-320nm) reaching the earth's surface. Recently an "ozone hole" has been discovered above the Qinghai-Tibet Plateau inChina. In order to examine the effects of the enhanced UV-B radiation on the growth and reproduction of the alpine species on Qinghai-Tibet Pleteau, we use wild legume forages-Vicia unijuga A as research materials in laboratory conditions with the UV-B radiation level was6.4K.J.m-2.d-1(simulating a9%ozone depletion over Gannan,China34。55'N,2900m). The growth and development, physiology and biochemistry, gene expression under UV-B radiation were explored in this study. The plant height, aboveground biomass, leaf area, endogenous hormones, plant nutrients, lignin synthesis-related gene expression levels and other indicators were measured. The results showed:
1) Enhanced UV-B radiation made the leafes of Vicia unijuga A skew, asymmetries, brown spots on the surface, and lignin deposition thickened in stems. In the central of stem, the medullary cavity was increase.
2) A short time of enhanced UV-B treatment (10days) had no significant effect in plant height and aboveground biomass of Vicia unijugaA, with the accumulation of UV-B treatment time and the amount of radiation, they was lower than that of the control group significantly. Early UV-B radiation treatment has a stronger inhibitory effect on the growth of root of Vicia unijuga A, in the later period, it was the root growth frist. And UV-B radiation reduced the leaves area of Vicia unijuga A significantly.
3) In10days of UV-B treatment, the plant endogenous hormones ZT,6-BA, GAs, IAA were higher than the control group significantly.10days later, the content of them were decreased rapidly and significantly lower than the control group with the irradiation time. Changes in the content of GAs and IAA were most dramatic. UV-B treatment increased the ABA content significantly.
4) Total nitrogen content was higher than that of the control group in the first10days, but with the time extension of UV-B treatment, the rate of decline of total nitrogen is faster than the control group, which indicated that UV-B irradiation accelerated the consumption of plant total nitrogen. The content of organic carbon was higher than the control group significantly in the first10days of UV-B irradiation treatment, after which, the content was no significant difference between the control group. The content of total phosphorus is consistent with the changes in the content of organic carbon, which showed that the content of organic carbon and nitrogen of Vicia unijuga A have a less affected by UV-B irradiation enhanced.
5) In the whole process of UV-B radiation, the express of CAD gene was lower than the control group, the expression is suppressed, it's more sensitive to UV-B radiation. Despite the express of F5H gene has an increase in the whole process of the UV-B, compared to the expression level of the control group, it's still suppressed. Gene expression of CCOAOMT in the whole process of UV-B radiation presents a rising trend, which shows that the gene is susceptible to UV-B radiation excited, and it is the key gene in lignin synthesis.
At the same time, we constructed a full-length cDNA Library of Vicia unijuga A by SMART technique. According to the evaluation on quality, The titer of primary library was2.3×106pfu·mL-1, while the amplified library has a high titer of6.2×109pfu·mL-1and92.7%recombinant percentage.16plaques were randomly picked from the amplified library, after amplified by PCR identification, the results showed that the inserted fragment is mostly distributed between500to2500bp. The3'end full-length cDNA of CAD, a Vicia unijuga A gene related with lignin synthesis cloned by RACE method in the past, was amplified in this cDNA Library by PCR, which indicated that the cDNA library for leaves of Vicia unijuga had been constructed successfully by using the SMART technique.
引文
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