Hbice Gene Family Cloning,Expression Analysis And Interaction Protein Screening

Plants often suffer from cold stress during growth and development. In the long-term evolutionary selection process, the plants develop an efficient and complex response mechanism to resist and adapt to cold stress. ICE(inducer of CBF expression)-mediated ABA-independent signal transduction pathway plays an important role in regulating the responses of temperate plants to cold stress. Little is known about this pathway in tropical plant。In the present study,ICE family members were isolated,their expression in bark tissues and rubber tree clones with differential cold-tolerance was analyzed, and the interaction among ICEs and the interaction of ICEs with JAZ members were investigated by using physiological, biochemical and molecular biological techniques. The main results as follows:1. Methyl jasmonate application significantly enhanced the cold tolerance of the cold-sensitive rubber tree clone Reken501 upon 4℃ treatment.2. The full-length cDNAs of 5 members of the ICE transcription factor family were cloned from the leaves and bark of rubber tree clone Reyan8-79 by RT-PCR. The were named as HbICE1, HbICE2, HbICE3, HbICE4 and HbICE5 with respective (Genbank accession numbers of KY406162, KY406163, KY406164, KY406165 and KY406166).Bioinformatics analysis showed that the five genes encoded the proteins with a typical bHLH domain, belonging to the ICE transcription factor family members.3. qPCR analysis was performed to show the expression pattern of the HbICEs and HbJAZs genes in latex, root, bark and leaf. HbICE1 and HbICE3 were mainly expressed in the leaves while HbICE4 and HbICE5 were mainly expression in bark tissuesof the highest.The high expression of HbICE2 was equally present in the leaves and bark tissues. It is noted that all the five ICE genes were little expressed in latex. The HbJAZ1, HbJAZ2,HbJAZ4, HbJAZ5 and HbJAZ6 were mainly expressed in the bark tissues while HbJAZ3 was mainly expressed in latex. The HbJAZ7 was equally expressed in abundance in latex,bark tissues and leaves.4. qPCR analysis showed that the expression level of HbICEs in the primary bark was significantly higher than that in the regenerated bark, and in the rubber tree clone PR107 with a relatively strong cold-tolerance was significantly higher than that in rubber tree clone Reyan8-79 with a relatively weak cold-tolerance. By contrast, the expression level of HbJAZ genes was significantly higher in regenerated bark tissues than that in the primary bark tissues. And moreover, their expression levels in the regenerated bark tissues of rubber tree clone Reyan8-79 were significantly higher than that of rubber tree clone PR107. Based on the expression pattern, HbICEs may be important in regulating the response of bark to cold stress.5. Yeast two hybrid showed that the HbICE5 could respectively interact with HbICE1,HbICE2 and HbICE3.6. Yeast two hybrid showed that there present interaction between HbICE3and HbJAZ1, and between HbICE5 with either HbJAZ1 or HbJAZ2.Conclusions:1. Jasmonate has effect on enhancing the cold tolerance of rubber tree to cold stress.2. The HbICE genes are mainly expressed in bark tissues and leaves, other than laticifers. Their expression levels are positively related to the ability of rubber tree to cold stress.3. The expression patterns of HbJAZ genes between the primary bark and regenerated bark, and between the rubber tree clone PR107 and Reyan8-79 with differential cold tolerance were reverse to those of HbICE genes and negatively related to the ability of rubber tree to cold stress.4. The HbICE3 and HbICE5 as well as HbJAZ1 and HbJAZ2 may be the key members in the JA signal transduction pathway of the response of Hevea bark tissues to cold stress.