Clone And Functional Veirfication Of Leidl And Lehaesa And Regulations Of Calmodulin Of Lemkks And Lempks In The Tomato Pedicel Abscission

Abscission is a physiological process that plant cell, tissue or organ separate from thematernal. The specific region abscission occurred at is called abscission zone. In tomatoproduction, especially in the facilities tomato cultivation in winter and spring, it easily causesflower and fruit dropping. Although some genes related to the abscission development havebeen isolated, less study about the signal transduction pathways of tomato pedicel abscissionhave been done. In this article, tomato pedicels were taken as test materials, cDNA sequencewas gained by RACE and the application of reverse genetics made detailed analysis on them.The main results were as follows:1. The primers were designed according to IDA and HAESA conserved regions. About400bp and700bp core sequence similar with other plants were obtained by homologousamplification. Then we gained the3’and5’ terminal by using RACE(cDNA ends rapidamplification technology). Finally510bp and3035bp were obtained by splice, named themLeIDL and LeHAESA Respectively. Bioinformatics analysis explain that: They have510bpand2667bp ORF respectively, appear high similarity with IDA, IDL（IDA-LIKE）, HAESA,HSL（HAESA-LIKE） as reported. Infering they encording101and888amino acids, withmolecular weights of11456.3and97874.1, isoelectric point of9.56and6.35repectively. Thetwo genes have expressed at pedicel abscission zone in different developmental stages of theflower by qRT-PCR analysis, but LeIDL was more stablely expressed during the wholeprocess. With the process of flower development, LeHAESA showed obvious regularity:while the petals had not open, its expression was positively correlated with its development.After flowering, it showed high expression level at early fruit set and then decreased until latematuring, it rose again. In different tissues of the flower, LeIDL expressed highest in petalsand stamens second. While LeHAESA expressed highest in pistil and followed by stamens.The results shows that they involved in both tomato pedicel abscission and flowersdevelopment.2. In this study,“Chinese vegetables6th” was taken as the test material to Investigate theinduced role of IAA and6-BA in callus and adventitious buds and NAA in adventitious roots.The results showed the best induced differentiation medium formula for “Chinese vegetables6th” is MS+0.2mg·L-1IAA+1.0mg·L-16-BA, and the best induced rooting medium formulais MS+0.05mg·L-1NAA.In this study, pMDC141was taken as overexpression vector. Inorder to find the transformed cells resistance against Hyp, we set six-gradient test as0mgL-1,5mgL-1,7mgL-1,9mgL-1,11mgL-1,13mgL-1. Once it reached9mgL-1, explant woundappeared brown until it completely dead. As a result,7mgL-1was used in the follow-up testsof adventitious buds resistance screening concentration, and eight negative control were obtained. At the same time, PB7GWIWG(II) was taken as RNAi expression vector toinvestigate the transformed cells resistance against PPT. In initial screening, we setsix-gradient as0mgL-1,0.5mgL-1,1.0mgL-1,1.5mgL-1,2.0mgL-1,2.5mgL-1and inlater screening, we set six-gradient as0mgL-1,0.6mgL-1,0.8mgL-1,1.0mgL-1,1.2mgL-11.4mgL-1PPT. When the concentration of PPT was1.0mgL-1, there was few budsdifferentiation, and moreover the buds turned yellow to bleaching gradually. Therefore,0.8mgL-1was used in the follow-up tests of adventitious buds resistance screeningconcentration.3Buliding expression vector of LeIDL and LeHAESA using Gateway technology. Thetransgenic plants were tested by PCR.The results found that the transcription was improvedby technology of overexpression, the transcription; was inhibited by RNAi. Plantinflorescence are added in35S: LeIDL; the development of fruit was anomaly in RNAi:LeIDL; floral organs anf fruit was unnormal in35S:LeHAESA4System phylogenetic tree showed that LeMKK2and AtMKK4/AtMKK5,LeMPK1,LeMPK2and AtMPK6, LeMPK3and AtMPK6are close relatives, suggesting that they havesimilar function. qRT-PCR analysis showed LeMKK2, LeMPK1, LeMPK2positively regulatepedicel abscission. Ca play an amplification role of the signal cascades of MAP in the earlytime of abscission. The treatment of Ca+W7restored or partly restored the inhibitory effect ofCa on LeMKK1,3,4gene expression, but exacerbated the inhibitory effect on LeMKK2andthe treatment completely restored the inhibition effect of Ca on LeMPK1.