Effect Of AtDWF4 Gene Overexpression On Growth, Development And Cold Resistance In Brassica Juncea

Plant hormones play an important role in plant growth and development process. Plant hormones have significant effects on plant growth, development, senescence and dormancy and stress-tolerance. Brassinosteroids are new plant hormones confirmed recently, that have been recognized to involve regulation of auxin, cytokinin, gibberellin, abscisic acid, ethylene as the sixth plant hormones, and to participate plant cell elongation, vascular differentiation, root growth, light reaction, stress-tolerance, senescence as so on.The phytosterol precursor is metabolized testosterone and brassinolide with biological activity through the complex parallel and interactive network of metabolic pathways. The Arabidopsis DWF4 gene encoding a cytochrome P450 oxidase, catalyze C-22 hydroxylation reaction of brassinosteroid biosynthesis. And the DWF4-mediated step represents a bottleneck and thus serves as a rate-determining step of the entire network of biosynthetic pathways. This determines the amount of final BR synthesis.Mustard(Brassica juncea Coss.) is one of the important cruciferous vegetable crops, cultivated in Southwest China and the Yangtze River Basin widely. DWF4 gene of Arabidopsis thaliana was overexpressed in stem mustard to study the effect the DWF4 gene expression on mustard plant growth, development and cold resistance ability, and to promote mustard biological yield, seed yield and cold hardiness ability. The main results obtained are as follows. 1. The generation of DWF4 transgenic mustard plantsThe expression vector pCABarDWF4 was transformed into mustard Via Agrobacterium mediated method. The pCABarDWF4 transgenic Brassica juncea plants were obtained through constant rounds screening by added 8 mg/L phosphinothricin(PPT) in culture medium. And further PCR results of transgenic plants indicated the DWF4 gene had been successfully integrated into the genome of Brassica juncea. 2. The pCABarDWF4 transgenic plants herbicide resistance identification and the DWF4 gene expression analysisThe PCABarDWF4 transgenic plants offspring and wildtype mustard seedlings were treated using concentration of 500 mg/L PPT, the results showed the progenies of PCABarDWF4 transgenic plants were highly resistant to herbicide. Three lines were used to analysis the DWF4 gene expression, and the expression levels were higher in D2 and D3 plants than D1 plant. 3. T2 pCABarDWF4 generation of transgenic mustard seedling growth in light and darkThe transgenic seedlings were obviously longer hypocotyl and root whether under light and dark conditions than wild-type controls. The results indicated the DWF4 gene overexpression can promote the growth and development of transgenic plants at the seedling stage. 4. T2 generation pCABarDWF4 transgenic seedling treated with BR inhibitor PCZPCABarDWF4 transgenic mustard seeds germinated on petri dish with 50 μmol PCZ brassinolide inhibitor. Compared with the wild-type control, the root length and hypocotyl length were significantly higher, the results showed that transgenic plants were more resistant to PCZ growth inhibition. The results further supported the DWF4 gene expression in plant cells can improve brassinosteroid biosynthesis and accumulation. 5. The growth, pod and seed investigation of T2 generation pCABarDWF4 transgenic mustard plantsThe effect of DWF4 overexpression on plant growth was monitored during the course of development. The PCABarDWF4 transgenic Brassica juncea plants displayed faster growth and stronger growth potential as compared to the wild-type control. The leaf, flower and other organs of transgenic plants are greater than the wild-type control. The plant height, expansion diameter, maximum leaf width, maximum leaf length was significantly higher than that of wild type. And the bolting time and flowering time of transgenic plants were earlier than wild-type. In addition, compared with wild-type,the pCABarDWF4 transgenic plant plants were found more fruiting branches and longer branches, pods and seeds. 6. Biosynthetic genes and transcriptional regulation genes expression analysis of BRThe expression of BR biosynthetic genes and transcription regulation related genes in transgenic plants were analyzed respectively. The results showed that the expression of CPD gene was significantly down regulated in transgenic plants, the expression levels of DET2, ROT3 and CYP85A2 were significantly higher than those in wild-type. And the expression analysis of transcriptional regulatory genes for BR biosynthesis showed that the expression level of BAK1(BRI1), BES1, BZR1, BIN2 and BSKs genes were higher than those in the in wild-type except for BAS1 gene. The results showed that the DWF4 gene overexpression affected both the expression of BR biosynthesis related structure gene and transcriptional regulation gene, leading to the level of endogenous BR raise and promote plant growth, more pods and seeds generation. 7. Auxin related gene expression analysis and auxin inhibitor TIBA treatment of T2 generation pCABarDWF4 transgenic mustard seedlingIn DWF4 transgenic plants, auxin synthesis genes ARF2 and ARF7 expression levels were significantly higher as compared with the wild type. And the transgenic seeds were germinated on petri dish with 5 μmol TIBA(an auxin inhibitor) in it, obviously weaker growth inhibition of root and hypocotyl of transgenic plants were observed than the wild type control, indicating that the overexpression of DWF4 lead to endogenous BR accumulation levels rise, promote the synthesis of endogenous auxin, reducing effect of auxin inhibitors TIBA to the growth of transgenic plants. 8. Cold tolerance of T2 generation pCABarDWF4 transgenic plantsThe root and hypocotyl of the T2 generation pCABarDWF4 transgenic seedlings were significantly length than that of the wild-type at 6 ℃. The transgenic plants and wild-type control were treated at 4 ℃ for 3 days, lower relative electrical conductivity were observed of transgenic plants than that of wild-type, while proline accumulation was significantly higher in transgenic plants than that of the wild-type control. In additional, the transgenic plants were recovery faster than wild-type plants after out of the low temperature stress. The results indicated DWF4 gene overexpression improved the cold resistance of plants. The further gene expression analysis showed that expression levels of cold resistance related gene CBF1-3, ICE1, HOS1, SIZ1, MAPK3, KIN1 were higher than the wild type.