Dissection Of Hormone Signaling Pathway In The Regulation Of Cold Stress Resistance In Rubber Tree (Hevea Brasiliensis Muell.arg)

Plant hormones play an important regulatory role in diverse plant defense responses and various developments.Jasmonic acid,abscisic acid,ethylene and auxin signaling pathways interact to regulate plant tolerance to cold stress.Rubber tree is a typical tropical economic crop.China’s rubber planting area is located in the northern edge of the world tropics.The frequent cold current attacks seriously threaten the sustainable development of natural rubber planting industry.The research on the cold resistance mechanism of rubber tree has important practical value for breeding new varieties with high yield and cold resistance and improving cold stress resistance of rubber tree by means of genetic engineering.In this study,Based on the established indoor identification and evaluation system of cold resistance of rubber tree,the effect of cold stress on jasmonate biosynthesis was investigated,candidate genes responding to cold stress were isolated and identified,their expression patterns were analyzed,and their functions were identified,and the relationship between target genes was further analyzed by using physiological,biochemical and molecular biological techniques,to analyze the jasmonic acid signal transduction pathway and its interaction with other hormoneson cold resistance of rubber tree.The main results as follows:1.A laboratory identification and evaluation system for cold stress resistance of rubber tree was established by using seedlings of 25 rubber tree clones with known cold resistance,which were treated at 4 ℃for 4 days and cultured for 20 days to identify the cold resistance.2.Under cold stress,the contents of JA and ABA in rubber tree increased,and the contents of auxin and ET in rubber tree decreased significantly.3.The full-length c DNAs of 66 genes from JA signaling pathway,23 genes from ABA signaling pathway,10 genes from ET signaling pathway,11 genes from IAA signaling pathway,and 7 Hb HSP genes were cloned from rubber tree by RT-PCR.and the cloned and identified genes structure and proteins structure were analyzed by software.4.q PCR results showned thatthe expression pattern of 84 JA related genes,23 ABA related genes,10 ET related genes,11 IAA related genes,7 Hb HSP genes,and 6Hb SIZ genes were analyzed in 93-114 and Reken 501 under cold stress.A total of 41 candidate genes responding to cold stress were screened.5.q PCR analysis showned thatthe expression levels of Hb ICE1,Hb ICE2,Hb ICE3,Hb ICE4,Hb DREB1,Hb DREB2,Hb DREB4 and Hb DREB22 in three cold resistant clones were significantly higher than those in three non cold resistant clones.6.By Agrobacterium mediated genetic transformation system,overexpression of Hb ICE1,Hb ICE2,Hb ICE4 and Hb ICE5 improved the cold resistance of transgenic Arabidopsis and tobacco,respectively.7.Hb DREB1,Hb DREB4 and Hb DREB22 conferred transgenic Arabidopsis by Agrobacterium tumefaciens enhanced tolerance against cold,respectively.All the proteins were located in the nucleus of onion epidermal cells,respectively.8.Using Agrobacterium mediated method,overexpression of Hb ARF2 and Hb ARF16.3 inhibited the growth of transgenic tobacco respectively,and Hb ARF2 also enhanced the cold resistance of transgenic tobacco.9.The results of yeast single hybrid showed that Hb MYC22,Hb MYC3,Hb ICE1,Hb ICE2 and Hb ICE3 could bind to the promoter of Hb DREB1,respectively,Hb MYC3 and Hb MYC25 could bind to the promoter of Hb Hsf A6 a,and Hb ICE4 could bind to the promoter of Hb DREB21.On the basis of the results above,the conclusions as follows:1.A efficient and accurate indoor evaluation system for cold resistance of rubber tree was established,which is a good technical platform for rapid,accurate and large-scale evaluation of cold resistance of rubber tree.2.JA,ABA,ET and IAA were involved in the cold stress response of rubber tree,and JA played an important role in the cold stress response of rubber tree.3.Four ICE genes and three DREB genes related to cold resistance were identified.It will lay a good foundation for developing cold resistance-related molecular markers and enhancing the cold resistance of high-yield rubber trees by transgenic technology.4.The JA signaling pathway mediated by Hb MYC-Hb DREB and Hb ICEHb DREB signaling pathways plays an important role in regulating the cold stress response of rubber tree.5.The Hb MYC3/25-Hb Hsf A6 a pathway integrated heat shock module HSF-HSP and JA signals to regulate the cold stress response of rubber tree.6.Hb ARF2 was a negative regulator of growth,and it could also enhance the cold resistance of transgenic plants,indicating that Hb ARF2 may be the interaction node between rubber tree growth and cold signal pathway.