Expression Profiles,Characterization And Fuction Of SHSP Gene Family In Hevea Brasiliensis

Hevea brasiliensis is not only the main source of natural rubber,but also the important strategic material related to the national economy,people’s livelihood and national defense security.Tapping panel dryness（TPD）of rubber tree is a complex physiological syndrome that severely restricts the production of natural rubber.The area for planting rubber trees in our country is very limited.As the contradiction between supply and demand of natural rubber becomes increasingly prominent,TPD has become one of the main factors that limit the output of rubber trees per unit area.However,the molecular mechanisms underlying TPD remain poorly understood.Our previous works suggested that the programmed cell death（PCD）caused by overdose of reactive oxygen species（ROS）was the key pathway,meanwhile small heat shock protein（sHSP）interacting with translationally controlled tumor protein（HbTCTP）,a key gene associated with TPD,may play important roles in scavenging ROS.In order to further clarify whether sHSP plays an important role in the process of TPD,In this work,sHSP,which interacts with HbTCTP,is used as a breakthrough to comprehensively compare and analyze the gene expression patterns of sHSP family members of rubber tree and the interaction between sHSP family members and HbTCTP.In addition,we focus on exploring the biological functions of small heat shock proteins that may be associated with TPD.The main results are as follows:1.According to the molecular weight of the 14 sHSPs in Hevea brasiliensis candidate gene family members,they were standardized and named HbsHSP15.8,HbsHSP16.2,HbsHSP17.3,HbsHSP17.4,HbsHSP17.5a,HbsHSP17.5b,HbsHSP17.5c,HbsHSP18.2,HbsHSP18.3,HbsHSP22.5,HbsHSP22.6,HbsHSP23.8,HbsHSP23.9,HbsHSP25.8,respectively.The results of bioinformatics analysis showed that all 14 sHSPs hadα-crystallin conserved domains.The secondary structure prediction results showed that the 12 sHSPs were all composed ofα-helix,β-turn,extended strand and a great quantity of random coils.The above two features are typical structural features of sHSP protein,and are important structural features for sHSP protein to perform its functions.2.HbsHSP15.8,HbsHSP16.2,HbsHSP17.3,HbsHSP17.4,HbsHSP17.5b,HbsHSP18.2,HbsHSP18.3,HbsHSP22.5,HbsHSP23.9,HbsHSP17.5c and HbTCTP were highly expressed in latex.In addition,HbsHSP17.4,HbsHSP17.5a and HbsHSP25.8 were highly expressed in bark,stem and leaves,respectively,while HbsHSP23.8 was highly expressed in latex and stem.Members of the sHSP family and HbTCTP were also expressed at different developmental stages of rubber tree leaves.Under ethylene treatment,the expression of HbTCTP gene showed an upward trend with the severity of dead skin disease.The expression patterns of HbsHSP16.2,HbsHSP17.3,HbsHSP22.5 and HbsHSP17.5c are similar to HbTCTP.The expression trends of HbsHSP15.8,HbsHSP17.4,HbsHSP17.5a,HbsHSP17.5b,HbsHSP18.2,HbsHSP18.3,HbsHSP22.6,HbsHSP23.8,HbsHSP23.9 and HbsHSP25.8 genes are opposite to HbTCTP.Therefore,HbTCTP and 14 sHSP genes can be regulated by ethylene.3.The self-activation experiment showed that HbTCTP has no self-activation activity.4.The results of the yeast two-hybrid experiment showed that HbTCTP did not interact with HbsHSP16.2,HbsHSP17.3,HbsHSP17.4,HbsHSP17.5a,HbsHSP17.5b,HbsHSP18.3,HbsHSP23.8,HbsHSP23.9 and HbsHSP25.8.The above nine rubber tree sHSP members may not participate in the TPD pathway.5.The real-time RT-PCR results showed that the expression levels of HbsSHP17.5c and HbTCTP in wild rubber tree varieties were mostly much higher than those in cultivated rubber tree varieties.6.The results of abiotic stress treatment showed that HbsHSP17.5c can significantly improve the ability of transformed strains to resist H₂O₂ stress,low temperature,high salt and metal ion stress.7.We constructed HbsHSP17.5c binary expression vector and transformed it into tobacco via Agrobaterium-mediated plant transformation.Currently,we have obtained 14positive transgenic plants overexpressing HbsHSP17.5c,which lays a solid foundation for further elucidating the function of HbsHSP17.5c.