Response Of Rate-limiting Enzyme Gene StCPD And StDWF4 Regulating Brassinosteroids Synthesis To Drought And Salt Stress In Potato

Brassinolide?BL?are a new type of plant growth regulators and are considered the sixth class of phytohormone because they were found in almost all the phyla of the plant kingdom.At present,more than 70 kinds of BL have been found in crops,and they were collectively referred to as Brassinosteroids?BRs?.CPD and DWF4 encodes C-3 oxidase and C-22 hydroxylase?cytochrome P450 enzyme?respectivrly,which plays a vital role in BRs biosynthesis?Normura and Bishop,2006;Bancos,et al.,2006?.To date,there are few reports on the alteration of endogenous BRs using their biosynthesis gene to enhance stress resistance at the molecular level.Thus,the aim of this study was to elucidate the role of CPD and DWF4 proteins in potato stress resistance and provide a foundation for improving plant tolerance against drought and salt stress and the role of the antioxidant defense system in this process.In this study,both CPD and DWF4 genes were isolated in the potato,then the corresponding over-expression vectors were constructed,and CPD interference expression vector pRS-CPD-Ri was constructed using the plant expression vector pRS-300.They were transferred into the potato genome,mediated by Agrobactium tumefacienses L.,and transgenic plants were obtained.Finally,the function of the CPD and DWF4 gene from potato was explored through morphology,molecular biological and physiological and biochemical analysis of the obtained transgenic plants and untransgenic plants?NT?.The main results are as follows:1.The full-length cDNA of StCPD and StDWF4 homologous with electronic cloning sequence of CPD and DWF4?GenBank accession XM₀06338792.1 and XM₀06340546.1?in NCBI,were amplified in potato,named StCPD and StDWF4 respectively.Bioinformatics regarding StCPD and StDWF4 were analyzed.The results showed as follows: the coding region of StCPD and StDWF4 was 1473 bp and 1476 bp,encoded a 490 and 491-amino acid polypeptide.The protein deduced from the StCPD and StDWF4 shared 77% and 74% aa indentity to CYP90A1 and CYP90B1 in Arabidopsis,were named StCYP90 A ? StCYP90 B.2.The full-length cDNA of StCPD and StDWF4 gene was linked into the pBI121 vector under the control of a medium constitutive promoter?CaMV 35 S promoter?and created a StCPD and StDWF4 gene overexpression vector?pBCPD and pBDWF4?.Slices of stem and microtubers of potato cultivar ‘Zihuabai' were used as the receptors for Agrobacterium-mediated transformation,and 11 CPD-over-expressed and five StDWF4-over-expressed transformed plants were obtained.The relative expression level of in different transgenic lines was analyzed using Quantitative real-time polymerase chain reaction?qRT-PCR?.The expression levels of all transgenic lines were higher than those of control?NT?,except for C10.The transcription level of StDWF4 in pBCPD transgenic lines was negatively correlated with that of StCPD.The transcription level of StCPD in pBDWF4 transgenic lines was positively correlated with that of StDWF4.The StCPD and StDWF4 was expressed in the roots,stems and leaves of plants,and with higher StCPD expression in the leaves and stems of the transgenic lines and NT plants than that in roots,but with higher StDWF4 expression in the roots and stems of the transgenic lines and NT plants than that in leaves.3.For each StCPD gene transgenic and NT plant sample after treatment with PEG,the relative expression of StCPD gene were determined by qRT-PCR,the physiological and biochemical indexes and the activities of antioxidant enzymes of drought treatment and NT plants were measured.The effects of drought stress on the relative expression of the StCPD gene in transgenic and NT plantlets were highly significantly different and increased with extension of the drought-treated time until 48 h.The relative expression levels of StCPD gene were higher in transgenic than in NT plants.In StCPD-overexpressing potato plants compared with NT plants under drought stress conditions,the contents of MDA were decreased,proline,soluble protein and soluble sugar increased.In plants exposed to drought for different times,the content of MDA was increased,and the treatment of 48 h decreased slightly;the proline soluble sugar levels were increased from 0 h to 48 h,soluble protein contents was a significant decline in both transgenic and NT seedlings with different PEG treatment times.When the treating time was prolonged,the activities of antioxidative enzymes?SOD,POD,CAT,GR and APX?exhibited increasing trends in response to drought stress until 48 h.Under drought stress,the growth performance of the StCPD transgenic plants was better than NT plants in quantitative estimation of the height of plantlets,the thickness of the stem,the roots length,fresh weight of plantlets,fresh weight and diameter of microtuber.4.Each StDWF4 gene transgenic and NT plants were treated with 200mmol/L NaCl for different treatment times?0 h,2 h,6 h,12 h,24 h and 48 h?,the relative expression of StDWF4 gene were determined,content of MDA,proline,soluble protein,soluble sugar and the activity of five antioxidant enzymes were measured.The relative expression levels of StDWF4 gene were higher in transgenic than in NT plants,which were increased with extension of the drought-treated time from 0-24 h.Under both drought and control treatments,the content of MDA in transgenic plants was significantly lower than NT,the MDA content is 1.18-1.60 times of transgenic plants,but proline,soluble protein,soluble sugar content of transgenic ones is 1.25-1.55,1.12-1.45,1.41-2.89 times than that of NT;the activity of SOD,POD,GR,CAT and APX in NT were 1.19-1.70,1.19-2.02,1.41-2.89,1.16-2.15,1.17-1.62 times compared of StDWF4 transgenic plants.Under both drought and control treatments,the variation trends of MDA,proline,soluble sugar,soluble protein content and SOD,POD,GR,CAT and APX enzyme activity in the StDWF4 gene transgenic and NT plants were consistent with the trends of StCPD over-expressed plants.Under salt stress,the growth performance of the StDWF4 transgenic plants was better than NT plants in index of the height of plantlets,the thickness of the stem,the roots length,fresh weight of plantlets and fresh weight and diameter of microtuber.5.The potato StCPD interference expression vector pRS-CPD-Ri was constructed using the plant expression vector pRS-300 and transferred into the potato genome,mediated by Agrobactium tumefacienses L.,and transgenic plants were obtained.The morphology,molecular biology and physiological and biochemical indexes of the transgenic lines were analyzed.The StCPD gene expression levels were analyzed by qRT-PCR,the results showed that the relative expression levels of five different transgenic lines were 0.22,0.52,0.10,0.32 and 0.43,respectively,which were significantly lower than those of NT.The relative expression levels of StCPD gene in PRS-CPD-Ri transgenic lines were leaves>stem>root.The plant height,stem diameter,root length and fresh weight of the transgenic plants?Ci1,Ci3 and Ci4?were significantly decreased compared with that of NT.6.Plant leaves were collected after different treatment times?0 d,2 d,4 d and 6 d?and used to measure the relative expression levels of StCPD gene and the content of MDA and proline.The relative expression level of StCPD gene in transgenic plants was significantly lower than that in NT,especially in drought treatment 6d,the former was 1.47 times of that of the latter,and the gene expression increased with the treatment time.Under drought stress,MDA content of transgenic potato plants was 1.07-1.17 times compared with NT,while the former is 1.02-1.29 times of the latter of control treatment;under control and drought treatment,the proline content of StCPD gene transgenic ones were 0.55-0.90 and 0.57-0.84 times of NT respectively.The plant height,stem diameter,root length,root length and fresh weight of the transgenic plants were significantly lower than that of NT plants after drought treatment of 15 d.The results might provide some useful insights that StCPD and StDWF4 gene may play important roles in drought and salt tolerance in potato.