毛果杨Rubisco活化酶基因的克隆与功能分析

发布时间：2018-08-18 18:30

【摘要】：【目的】核酮糖-1,5-二磷酸羧化酶(Rubisco)是参与植物光合作用的第1步碳同化的关键酶,而Rubisco活化酶(RCA)能够使Rubisco处于稳定的催化活性状态,从而提高光合效率。本研究从毛果杨中克隆RCA基因并通过遗传转化南林895杨,获得PtRCA高表达的转基因株系,并进行分子检测及相关功能分析,为培育杨树新型高光效抗逆品种提供依据。【方法】基于毛果杨基因组数据库信息克隆PtRCA基因序列,利用生物信息学对PtRCA基因进行功能和结构分析。采用Gateway技术将PtRCA构建到植物表达载体pGWB406中,以南林895杨为受体材料,通过农杆菌介导法进行遗传转化。以转PtRCA基因南林895杨和对照(南林895杨)为材料,测定分析在高温胁迫下转基因植株的基因表达、光合参数和叶绿素荧光参数的差异。【结果】从毛果杨中克隆获得PtRCA的CDS序列长1 323 bp,编码440个氨基酸残基,其蛋白相对分子质量为48 315.9 Da,等电点pI为5.57,为疏水性蛋白,无信号肽以及跨膜结构;通过序列对比,发现PtRCA属AAA+超级家族一员,且与大豆、拟南芥RCA蛋白同源性较高。转PtRCA基因南林895杨RCA表达量均高于对照,且能够利用强光充分进行光合作用,其光饱和点也均高于对照,增加约12.5%~37.5%;光补偿点除3号株系外,其他转基因株系均略低于对照;转基因植株在光饱和点的光合速率比对照增高24.6%~55.7%。转基因株系对CO_2的利用能力和羧化效率均强于对照组,CO_2饱和点除1号和4号株系外,其他株系均比对照低12.5%~25.0%;CO_2补偿点比对照降低53.1%~80.4%;光呼吸比对照低37.7%~79.3%,并且转基因杨树在CO_2饱和点的光合速率比对照增高4.4%~26.4%。另外,转基因杨树表现出耐光氧化的能力,光氧化处理后,对照PSⅡ原初光化学效率下降61.7%,而转基因杨树下降45.0%~53.1%;对照PSⅡ实际光化学效率下降54.1%,而转基因杨树下降38.7%~52.0%;光化学猝灭系数对照下降68.3%,而转基因杨树下降51.0%~65.8%;非光化学猝灭系数对照仅增加3.0%,而转基因杨树增加6.0%~26.5%。【结论】杨树Rubisco活化酶(PtRCA)蛋白与大豆、拟南芥RCA蛋白同源性较高。通过实时定量及相关生理分析表明,转PtRCA基因南林895杨具耐高温特性,利用CO_2和强光进行光合作用的能力较强,催化Ru BP进行羧化反应的效率较高。转PtRCA基因杨树能够充分利用吸收的光子,PSⅡ反应中心效率较高,过剩光能量得到较好的耗散,表现出耐光氧化的能力。研究结果表明,PtRCA基因的高效表达提高了转基因植株的光合效率,并对高温高光强具有调节能力。
[Abstract]:[objective] (Rubisco) is the key enzyme of carbon assimilation in the first step of plant photosynthesis, and Rubisco activator (RCA) can make Rubisco in a stable state of catalytic activity, thus improving photosynthetic efficiency. In this study, RCA gene was cloned from Populus pubescens and transformed into Poplar Nanlin 895 by genetic transformation. Transgenic lines with high PtRCA expression were obtained, and molecular detection and related functional analysis were carried out. [methods] PtRCA gene sequence was cloned based on genomic database of Populus tomentosa. Bioinformatics was used to analyze the function and structure of PtRCA gene. PtRCA was constructed into plant expression vector pGWB406 by Gateway technique. Poplar Nanlin 895 was used as receptor material for genetic transformation by Agrobacterium tumefaciens. The gene expression of transgenic plants under high temperature stress was determined and analyzed by using transgenic poplar 895 with PtRCA gene and control (Poplar Nanlin 895). [results] the length of CDS sequence of PtRCA was 1 323 BP, encoding 440 amino acid residues, the relative molecular weight of the protein was 48 315.9 DaA, and the isoelectric point Pi was 5. 57, which was hydrophobic protein. PtRCA belongs to AAA superfamily and has high homology with soybean and Arabidopsis RCA protein. The RCA expression of transgenic poplar Nanlin 895 with PtRCA gene was higher than that of the control, and the light saturation point was also higher than that of the control, and the light saturation point was also higher than that of the control, and the light compensation point was slightly lower than that of the control, except for line 3, the light compensation point was slightly lower than that of the control, and the light saturation point was also higher than that of the control. The photosynthetic rate of transgenic plants at the light saturation point was 24. 6% and 55. 7% higher than that of the control. The ability of CO_2 utilization and carboxylation efficiency of transgenic lines were higher than those of control group except lines 1 and 4. All the other lines were lower than the control at the compensation point of 25.0 and 53.1% lower than the control, and the light respiration was 37.7% lower than that of the control, and the photosynthetic rate of the transgenic poplar at the saturation point of CO_2 was 4.40.26.4% higher than that of the control. In addition, transgenic poplar showed the ability to resist photooxidation. The primary photochemical efficiency of control PS 鈪，

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