【摘要】：【目的】核酮糖-1,5-二磷酸羧化酶(Rubisco)是參與植物光合作用的第1步碳同化的關(guān)鍵酶,而Rubisco活化酶(RCA)能夠使Rubisco處于穩(wěn)定的催化活性狀態(tài),從而提高光合效率。本研究從毛果楊中克隆RCA基因并通過遺傳轉(zhuǎn)化南林895楊,獲得PtRCA高表達(dá)的轉(zhuǎn)基因株系,并進(jìn)行分子檢測及相關(guān)功能分析,為培育楊樹新型高光效抗逆品種提供依據(jù)。【方法】基于毛果楊基因組數(shù)據(jù)庫信息克隆PtRCA基因序列,利用生物信息學(xué)對PtRCA基因進(jìn)行功能和結(jié)構(gòu)分析。采用Gateway技術(shù)將PtRCA構(gòu)建到植物表達(dá)載體pGWB406中,以南林895楊為受體材料,通過農(nóng)桿菌介導(dǎo)法進(jìn)行遺傳轉(zhuǎn)化。以轉(zhuǎn)PtRCA基因南林895楊和對照(南林895楊)為材料,測定分析在高溫脅迫下轉(zhuǎn)基因植株的基因表達(dá)、光合參數(shù)和葉綠素?zé)晒鈪?shù)的差異�！窘Y(jié)果】從毛果楊中克隆獲得PtRCA的CDS序列長1 323 bp,編碼440個氨基酸殘基,其蛋白相對分子質(zhì)量為48 315.9 Da,等電點(diǎn)pI為5.57,為疏水性蛋白,無信號肽以及跨膜結(jié)構(gòu);通過序列對比,發(fā)現(xiàn)PtRCA屬AAA+超級家族一員,且與大豆、擬南芥RCA蛋白同源性較高。轉(zhuǎn)PtRCA基因南林895楊RCA表達(dá)量均高于對照,且能夠利用強(qiáng)光充分進(jìn)行光合作用,其光飽和點(diǎn)也均高于對照,增加約12.5%~37.5%;光補(bǔ)償點(diǎn)除3號株系外,其他轉(zhuǎn)基因株系均略低于對照;轉(zhuǎn)基因植株在光飽和點(diǎn)的光合速率比對照增高24.6%~55.7%。轉(zhuǎn)基因株系對CO_2的利用能力和羧化效率均強(qiáng)于對照組,CO_2飽和點(diǎn)除1號和4號株系外,其他株系均比對照低12.5%~25.0%;CO_2補(bǔ)償點(diǎn)比對照降低53.1%~80.4%;光呼吸比對照低37.7%~79.3%,并且轉(zhuǎn)基因楊樹在CO_2飽和點(diǎn)的光合速率比對照增高4.4%~26.4%。另外,轉(zhuǎn)基因楊樹表現(xiàn)出耐光氧化的能力,光氧化處理后,對照PSⅡ原初光化學(xué)效率下降61.7%,而轉(zhuǎn)基因楊樹下降45.0%~53.1%;對照PSⅡ?qū)嶋H光化學(xué)效率下降54.1%,而轉(zhuǎn)基因楊樹下降38.7%~52.0%;光化學(xué)猝滅系數(shù)對照下降68.3%,而轉(zhuǎn)基因楊樹下降51.0%~65.8%;非光化學(xué)猝滅系數(shù)對照僅增加3.0%,而轉(zhuǎn)基因楊樹增加6.0%~26.5%。【結(jié)論】楊樹Rubisco活化酶(PtRCA)蛋白與大豆、擬南芥RCA蛋白同源性較高。通過實(shí)時定量及相關(guān)生理分析表明,轉(zhuǎn)PtRCA基因南林895楊具耐高溫特性,利用CO_2和強(qiáng)光進(jìn)行光合作用的能力較強(qiáng),催化Ru BP進(jìn)行羧化反應(yīng)的效率較高。轉(zhuǎn)PtRCA基因楊樹能夠充分利用吸收的光子,PSⅡ反應(yīng)中心效率較高,過剩光能量得到較好的耗散,表現(xiàn)出耐光氧化的能力。研究結(jié)果表明,PtRCA基因的高效表達(dá)提高了轉(zhuǎn)基因植株的光合效率,并對高溫高光強(qiáng)具有調(diào)節(jié)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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