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。
[ 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 the No. 3 strain. Besides, the usage of COs and the carboxylation efficiency of the transgenic poplars were better. Expect No. 1 and No. 4 strains, CO2 saturation point was around 12.5% -25.0% less than control plants. CO2 compensation point and light respiration were proved to be 53.1% - 80.4% and 37.7% - 79.3% lower compared to the controls respectively. Moreover, the efficiency of photosynthesis at the CO2 saturation point was around 4.4% - 26.4% higher. The tolerance of transgenic poplars to light oxidation was increased according to this research. Under the light oxidation treatment, the primary photochemical efficiency of PS Ⅱ decreased 61.7% in control lines. And transgenic poplar decreased by 45.0% - 53.1%. The actual photochemical efficiency of PS Ⅱ in controls decreased 54. 1%. And transgenic poplar decreased by 38.7% - 52.0%. Photochemical quenching coefficient dropped 68.3 % , and 51.0% - 65.8% declined in transgenic poplars. Non photochemical quenching coefficient increased by 3.0% compared to the control, and 6.0% -26.5% increased in transgenic poplars. [ Conclusion] Poplar Rubisco activase （PtRCA） protein and soy protein, Arabidopsis thaliana RCA homology is higher. According to the real-time quantitative PCR and related physiological analysis, transgenic poplars （P. deltoides X P. euramericana ＇ Nanlin895 ＇ ） showed high-temperature resistance, higher efficiency of photosynthesis, increased ability to use CO2 and better catalyzing effect on carboxylation reaction. The efficiency of assimilating photons and supplying to PS Ⅱ was highly developed, and as a result, the light oxidation ability of transgenic poplars was proved to be enhanced. The efficiency of photosynthesis was improved by high expression of PtRAC gene in transgenic poplars, which also showed ability to adjust for high temperature and strong light.
出处
《林业科学》
EI
CAS
CSCD
北大核心
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)
