| More than98%of natural rubber in the world come from Hevea brasiliensis. Natural rubber is a renewable important strategic material and industrial raw materials.Its comprehensive performance is obviously better than the synthetic rubber, in some areas, such as aviation, aerospace and heavy-duty truck manufacturing and so on,natural rubber is irreplaceable. The domestication time of rubber trees from wild to cultivated is very short, its variety improvement has great potential. The existing main problems of the breeding of rubber trees are:(1)the genetic basis of existing cultivars is narrow and has high heterozygosity. The majority of existing cultivars in the world derived from the46wild rubber trees which had been imported from Brazil to Southeast Asia more than100years ago. We had taken advantage of their improved individual or the improved individual of hybrid F1to form cultivars by budding into clones and also popularization and application. So the genetic basis of cultivars is narrow and has high heterozygosity, especially a large number of wild germplasm which had been collected in1981.The problems facing now is how to speed up the innovation utilization of these germplasm.(2)the Characters of yield components were difficult to be analyzed,and the cross-breeding cycle was very long. Unlike the yield formation and harvest way of most economic crop, natural rubber has been synthesized and accumulated in laticifer cells and also has been obtained by constantly tapping. So the Characters of yield components were difficult to be analyzed.Lacking of effective technical means which could make an early decision for the Characters of yield components,which is the main reason resulting in up to more than40years of the cross-breeding cycle.The research of the molecular mechanism of yield formation, which will provide the theoretical basis for establishing the early selection technology of yield traits.Although we have known that the biosynthesis of natural rubber was a typical mevalonic acid(MVA) pathway, it has been little known that how to regulate the biosynthesis of natural rubber. Recently, the related family genes of the three core components of jasmonate signaling pathway have been cloned in laticifer cells, and preliminary evidence that the natural rubber biosynthesis in laticifer cells was mainly regulated by jasmonate signaling pathway. In this study, by means of biochemical and molecular biological techniques, we investigated the expression differencesof the key links gene HbJAZ2, HbJAZ3, HblMYCl, HblMYC3in jasmonate signaling pathway between high-yield and low-yield mature trees, mature trees and saplings and the expressions of HblMYCl and HblMYC3in hybrids in order to provide a good foundation for developing the yield associated molecular markers. We identified that MYC transcription factors of JAZ genes family in order to provide a good foundation for researching the regulatory mechanism of the jasmonate signaling pathway. And we take HbJAZ3gene as example to build prokaryotic expression system of HbJAZ3protein and HbJAZ3mutant protein in order to provide a good foundation for identifying the three core components of jasmonate signaling pathway. The results as follows:(1) The amount of the expression of HbJAZ2, HbJAZ3, HblMYCl, HblMYC3in latex in high-yield mature trees were higher than in low-yield mature trees;(2) The expression levels of HbJAZ2, HbJAZ3, HblMYCl, HblMYC3in latex between high-yield mature trees and youngs were roughly consistent, in particular, the expression levels of HblMYCl and HblMYC3perfectly;(3) The expression levels of HblMYCl and HblMYC3in latax of two serial numbers100and90in progenies of crossing combination78-35*PB5/51were highest, and the expression levels ofthe two genesin latex ofat least two of three serial numbers41,48,60in progenies of crossing combination were higher;(4) We have screened and identied transcription factors of HbJAZl、HbJAZ2and HbJAZ5from the cDNA library by yeast one-hybridization,including the MYC transcription factors, bHLH transcription factors, HMGB transcription factors, zinc finger domain transcription factors and leucine zipper domain transcription factors and so on;(5) The one to one yeast one-hybrid experiment illustrated that Hb1MYC1、 Hb1MYC2、HblMYC3、Hb1MYC4and Hb1MYC5were transcription factors of HbJAZ1and HbJAZ2, and Hb1MYC1、Hb1MYC2、Hb1MYC3and Hb1MYC4were transcription factors of HbJAZ5;(6) Taking HbJAZ3gene as example, theprokaryotic expression vector pET28a(+)-JAZ3, pET28a(+)-JAZ3-ZIM-mut and pET28a(+)-JAZ3-Jas-mut were successfully constructedby site-directed mutagenesis and prokaryotic expression technology, and transformed into E.coli Rosetta(DE3). The fusion protein could be induced to express by lmmol/L IPTG, in the form of inclusion body at37℃for2hours. Finally, we have got the JAZ3and mutant fusion proteins by His tag purification. The mutant proteins were introduced deletions of TIFY motif for the ZIM domain and the conserved amino acid FLEKRK for the Jas domain. On the basis of the results above, the conclusions as follows:(1) The expression levels of HbJAZ2, HbJAZ3, HblMYCl, HblMYC3in latex could be uesd to early prediction of rubber producing capacity;(2) In addition to the MYC transcription factors, there are other types of transcription factors which can regulate the expression of HbJAZ genes in the key links of jasmonate signaling pathway in laticifer cells;(3) HbJAZ proteins could be interact with a variety of MYC transcription factors. It suggested that the regulation of the biosynthesis of natural rubber by jasmonate signaling mightexist diversity.(4) Prokaryotic expression system of HbJAZ3protein and HbJAZ3mutant proteinwere successfully constructedby site-directed mutagenesis and prokaryotic expression technology, which provide a good foundation for the study of the characters of HbJAZ proteins domain and interaction with other proteins.It is of great importance to further studying the key links of jasmonate signaling pathway;... |
PDF Full Text Request