毛果杨Rubisco活化酶基因的克隆与功能分析  

Cloning and Functional Analysis of Rubisco Activase Gene from Populus trichocarpa

作　　者：尹吴[1] 孙伟博[1] 周燕[1] 诸葛强[1] 

机构地区：[1]南京林业大学南方现代林业协同创新中心林木遗传与生物技术省部共建教育部重点实验室,南京210037

出　　处：《林业科学》2017年第4期83-95,共13页Scientia Silvae Sinicae

基　　金：国家科技部"863计划"课题(2013AA102703);国家国际科技合作专项(2014DFG32440);国家自然科学基金项目(31570650);江苏省高校优势学科建设工程项目(PAPD)

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�[ Objective] Ribulose-1,5-bishosphate （Rubisco） is the key enzymes in the first step of carbon assimilation involved in plant photosynthesis. Rubisco was kept in a catalytic active state under the control of Rubisco activase （RCA） , as a result, the efficiency of photosynthesis will be improved. In this research, the PtRCA gene of poplars was cloned. Transgenic poplars with high expression of PtRCA were obtained through this research. As a result, a new type of high efficiency photosynthesis with stress tolerance was proved to be feasible through the analysis of molecular detection and functional analysis. [ Method] According to the sequence of PtRCA gene cloned from Populus trichocarpa, the analysis of function and structure of PtRCA were conducted through Bioinformatics software. PtRCA was constructed into the expression vector pGWB406 by using Agrobacterium mediated method. ＇ Nanlin895 ＇ poplar plants （P. deltoides ~ P. euramericana ＇ Nanlin895＇ ） were used in this research. Gene expression of both transgenic poplars and ＇ Nanlin895 ＇ plants were compared under high temperature stress. The parameters of photosynthesis and chlorophyll fluorescence were compared between them as well. [ Result] The CDS sequence of PtRCA was 1 323 bp. There were 440 amino acid residues, and protein molecular weight is 48 315.9 Da. The isoelectric point is 5.57, which is hydrophobic protein without signal peptide and membrane structure. PtRCA is proved to be in the same AAA ＋ family according to the result of sequence comparison. It has same RCA homology protein with soybean and arabidopsis as well. The expression quantity of transgenic poplars were higher than ＇ Nanlin895 ＇ in average. Transgenic poplars showed advantage of photosynthesis during the noon, the light saturation point was 12.5% to 37.5% higher. Moreover, the efficiency of photosynthesis was 24.6% to 55.7% higher than the plants used as control. However, the light compensation point is lower than that of plants without transgenosis expect

关 键 词：杨树 Rubisco活化酶基因 转基因 光合作用 

分 类 号：S718.46[农业科学—林学] S718.43