| Environmental stresses, such as high temperature,low temperature,drought, as well aspathogen and pest attacks, have adverse effects on plant growth and development. Accordingly,the plant also formed a complicated and effective mechanism of resistance to reduce the adverseeffects through long-term evolution. These mechanisms include stress perception, signaltransduction, regulation of gene expression and the corresponding metabolism, physiological andmorphological regulation. Regulation of gene expression is one of the key mechanisms and it isvery important to clarify the mechanism of stress resistance in plants and improve the geneticimprovement of plant anti-adversity. Pepper (Capsicum annuum L.) is a kind of importanteconomic crop and industrial processing raw material crops, but biotic and abiotic stresses haveseriously negative effects on pepper yield and quality. Illumination on molecular mechanism ofdisease resistance in pepper is helpful to combine modern biotechnology and conventionalbreeding. Furthermore, it is the important basic research for genetic improvement of stressresistance in pepper. Therefore, AP2/ERF transcription factor, two of pepper defense responsegenes was studied. The other defense-related genes such as lipid transfer protein and Arf-GAPsgene were isolated. The main results are as follows:1,Four clones, CaERF14,CaDREB3,CaAGD8and CaLTP1, were isolated from peppercDNA library, which complete cds were1572bp,1997bp,1938bp and1211bp, respectively. Thededuced amino acids contain283,357,408and129amino acid residues. A highly conservedAP2/ERF domain of ERF subfamily was found in CaERF14and CaDREB3proteins. Homologycomparison and molecular phylogenetic tree analysis showed that CaERF14belonged to the â…©a(B-4)subgroup of ERF subfamilyï¼›CaDREB3belonged to the â… b(A-6)subgroup of ERFsubfamily. There was a conserved ARF-GAP domain in CaAGD8clone, and an AAI domain inCaLTP1clone that showed CaLTP1was type-I nonspecific lipid transfer protein.35S::CaERF14-GFP fusion protein was found localized in the nucleus and cytomembrane,whereas35S:: CaDREB3-GFP was found localized in nucleus by using subcellular localizationanalysis in onion epidermal cells. Transient expression analysis showed that both CaERF14andCaDREB3proteins could bind the GCC-box but could not bind CRT/DRE motif. 2, The expression of CaERF14was induced up-regulated by different plant growthsubstances such as ET, SA,JA,ABA treatment and some environmental factors such as drought,salt stress and Ralstonia solanacearum inoculation. But it wasn't change by auxin treatment, heatand cold stress. The expression of CaDREB3was up-regulated under SA,JA,ET treatment,Ralstonia solanacearum and cold treatment. But it wasn't change by auxin treatment, salt stress,drought and heat. In addition,the expression of CaDREB3was down-regulated except at6h afterwound treatment.3,The germination rate of tobacco seeds with CaERF14overexpressed was higher thancontrol (wild-type tobacco) under drought and high salt stress; and the seedlings of T1generationgrew better as well. The fresh weight, chlorophyll content, proline content, the activities of SODand POD enzyme of transgenic plant were higher than that of control. There was no significantdifference between the T1generation plant and the control after Ralstonia solanacearuminoculation.The germination rate of tobacco seeds with CaDREB3overexpressed was higher than control(wild-type tobacco) under high salt stress. But the fresh weight, chlorophyll content, prolinecontent, the activities of SOD, CAT and POD enzyme of CaDREB3overexpression plant werelower than that of control. The T1generation plant showed the stronger resistance after Ralstoniasolanacearum inoculation.4,The transcript level of drought-related and salt-related genes such as NtERD10a,NtERD10b, NtERD10d, NtCA, NtSOD, NtTsip1were up-regulated in the CaERF14overexpression plants. However, the transcript levels of PR genes were lower than control. Thisindicated that the CaERF14overexpression plants were sensitive to Ralstonia solanacearuminoculation. The transcript levels of NtPR1,NtPR2,NtPR3,NtPR4genes were up-regulated byCaDREB3overexpression after Ralstonia solanacearum inoculation.In summary,the CaERF14gene was involved in a series of defense response gene expressionand CaERF14overexpression tobacco improved the tolerance against salt and drought stress. Theexpression of CaERF14was induced by ABA treatment, which deduced that CaERF14gene mayresponse to abiotic stress by ABA pathway. CaDERB3gene involved in the expression ofpathogenesis-related gene (PR), the CaDREB3overexpression tobacco showed salt-sensitivity andresistant to Ralstonia solanacearum inoculation. The ET SA and JA treatment induced the CaDREB3expression which showed that CaDREB3gene may be involved in the ET, or the SA/JA signal pathways. Bioinformatics and tissue specificity analysis showed that CaAGD8andCaLTP1may be involved in response plant biotic and abiotic stress. |
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