Cloning,Expression And Function Analysis Of Thioredoxin Gene HbTRX11 From Hevea Brasiliensis

Hevea brasiliensis is an important tropical cash crop.And it is the main source of natural rubber,an important strategic resource.China is a non-traditional gum planting area,and rubber trees often suffer from drought,low temperature,typhoon and other abiotic stresses during their growth.At the same time,the incidence of tapping panel dryness of rubber trees in rubber gardens is also increasing.All these seriously restrict the healthy and sustainable development of China’s natural rubber industry.Both abiotic stress and tapping panel dryness can break the balance of redox state of plant cells.Thioredoxin（TRX）,as an important component of the thioredoxin system in vivo,plays an important role in maintaining the balance of redox state and resisting oxidative stress in vivo.In the early stage,the research group identified the thioredoxin family genes of Hevea brasiliensis,and analyzed their expression in both healthy and dead Hevea brasiliensis latex.It was found that the expression of HbTRX11 gene in tapping panel dryness Hevea brasiliensis latex was significantly reduced.In order to analyze the biological function of HbTRX11 in Hevea brasiliensis,HbTRX11 was cloned by RT-PCR method,and bioinformatics analysis was carried out.Real-time quantitative PCR was used to analyze the expression of HbTRX11 in different tissues of Hevea tree,as well as the expression changes after treatment with various hormones（ethephon/ETH,methyl jasmonate/Me JA,salicylic acid/SA and abscisic acid/ABA）and abiotic stress（low temperature,drought,high salt,methyloviperin/MV and H₂O₂）.The subcellular localization of HbTRX11 protein was observed by transient transformation tobacco.The function of HbTRX11 in oxidative stress response was analyzed by yeast expression system.The main research results are as follows:（1）The thioredoxin gene HbTRX11 was cloned from rubber tree,and the length of open reading frame is 357 bp,encoding 118 amino acids.HbTRX11 protein contains TRX conserved domain and has highly conserved Cys-Gly-Pro-Cys active site,which belongs to the h-type TRX family of proteins.（2）HbTRX11 gene was expressed in latex,bark,roots,leaves,female flowers,male flowers and new shoots of rubber trees,and the expression in latex was significantly higher than that in other tissues.Compared with healthy trees,the expression of HbTRX11 in rubber latex of tapping panel dryness was significantly decreased,and the expression of HbTRX11 gradually increased to the normal level during the process of tapping panel dryness recovery.The expression of HbTRX11 gene was regulated by hormones such as ETH,Me JA,SA and ABA.The expression of HbTRX11 gene changed irregularly after ETH and Me JA treatment.SA and ABA treatment could significantly inhibit the expression of HbTRX11.The expression of HbTRX11 gene was regulated by abiotic stress such as low temperature,drought,high salinity,MV and H₂O₂.Low temperature,drought and high salt stress can obviously induce the expression of HbTRX11.MV and H₂O₂ oxidative stress can significantly inhibit the expression of HbTRX11.HbTRX11 gene may play an important role in tapping panel dryness and abiotic stress response of rubber tree.（3）The subcellular localization vector p CAMBIA1300-HbTRX11-GFP was constructed.The results showed that HbTRX11 protein was localized in the nucleus,cell membrane and cytoplasm of tobacco by Agrobacterium tumefaciens injection into tobacco lower epidermis and PEG-mediated protoplasts transformation.（4）The plant overexpression vector p CAMBIA1301-HbTRX11 was constructed.Which laid a foundation for the identification of the function of HbTRX11 gene by plant genetic transformation.（5）The yeast expression vector p YES2-HbTRX11 was constructed and transferred into Saccharomyces cerevisiae INVSc1.The transgenic yeast and non-transgenic yeast were treated with H₂O₂-induced oxidative stress.Discovered the expression of HbTRX11gene reduced the resistance of recombinant yeast to oxidative stress.Indicating that HbTRX11 gene is a negative regulator of oxidative stress resistance.In summary,HbTRX11 gene was cloned in this study,and HbTRX11 protein belongs to h-type TRX.The expression of HbTRX11 gene was significantly up-regulated in latex and down-regulated in tapping panel dryness latex,which was regulated by hormone and abiotic stress.HbTRX11 protein is localized in the nucleus,cell membrane,and cytoplasm.HbTRX11 gene is a negative regulator of oxidative stress resistance.