Cloning And Expression Of RCA Gene In Carya Cathayensis Sarg. And Its Function During Heat Stress

Hickory(Carya cathayensis Sarg.)was an important woody oil crop,its production was easily affected by natural stress factors,especially the high temperature.High temperature severely limited photosynthesis in hickory,which affected plant growth,fruit setting,organic accumulation and so on.Therefore,it was with great significance to exploit and utilize the genes related to photosynthesis in hickory.In higher plant,the key step of photosynthesis that CO2 fixation process needed catalysis of Rubisco enzyme,Rubisco enzyme activity was regulated by complex mechanism in plants.Numbers of studies had confirmed that Rubisco activase(RCA)could activate and maintain catalytic activity of Rubisco enzyme.Meantime,RCA was also considered as the key control factor of photosynthesis in various environmental stresses in plants.Therefore,it played an important role in strengthening Rubisco enzyme activity and stabilizing photosynthesis system,and further improving the resistance ability of plants to study function of RCA.RACE technique was used to clone cDNA sequence of 2 RCA genes,which named CcRCA αand CcRCAβrespectively.Lengths of open reading frame were 1419 bp and 1302 bp respectively in sequences,which contained 472 and 433 amino acids.DNAMAN software was applied for multiple sequence alignment of amino acid sequences of RCA genes between hickory with species such as grape and so on,the result showed that the similarity of amino acid sequences is very high,and the homology with species like grape was more than 87%.One another cDNA sequence of RCA in hickory was obtained in this study,named CcRCAβ2.The analysis shows that the similarity of CcRCAβ2 with CcRCAβin amino acid sequence was as highly as 96%,both of them belonged to the β-isoform of RCA genes.In addition,the vector pET28a+ was used to construct prokaryotic expression vector that carrying CcRCAα.Expression strains BL21(DE3)were induced by IPTG to express soluble heterologous protein of RCA,and optimal conditions were achieved through optimizing a series of conditions for prokaryotic expression:20 ml medium with 1 mm IPTG and induced at 28 ℃ for 4 to 6 hours,which laid a foundation for further study of RCA gene function in hickory.The expression patterns of RCA gene under high temperature stress was also studied in this paper,the results showed that,photosynthetic rate,Rubisco enzyme activity,RCA gene transcription and translation level declined when hickory under high temperature,limition of photosynthesis by high temperature,may be affected by RCA gene transcription,translation and lead to the change of RCA enzyme content.In further study found that grafting hickory was better than no-grafting hickory in ability of resistance to heat stress,photosynthetic rate and Rubisco enzyme activity of H-L variety(hunan hickory as scion)in 41 ℃ dropped as less as 12.8% and 19.9% respectively than other varieties.In addition to scion variety factor,M-L variety(American hickory as scion)showed good resistance to heat stress maybe associate with expression regulation of CcRCAβ2 gene.All above results declared that hickory RCA genes play an important role in heat stress,which provided the basis for further study in the relationship between RCA genes and the resistance of plants.