Cloning And Characterization Of Transcription Factors In Brassica Campestris Ssp. Chinensis And Functional Analysis Of Heat And Cold-regulated Proteins Involved In Abiotic Stress

Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity, and oxidative stress, are serious threats to agriculture and result in the deterioration of the environment. Plants need to response to multiple stresses and developmental signaling during the growth and development. Thus the expression of functional genes should be accurately adjusted and controlled. Many signal transduction pathways are activated and plenty of genes involved in abiotic stress response are induced to express in plants. At present, a number of transcription factor families have been found involving in plant stress responses, and each contain a different type of DNA-binding domain, including NAC, Myb, WRKY, and bZIP and so on.In this paper, a novel NAC type transcription factor, nambed BrNAC, was isolated using rapid amplification of cDNA ends (RACE) method from Brassica campestris ssp.chinensis. The full-length cDNA of BrNAC has an 837bp open reading frame (ORF) and encode a NAC subgroup transcription factor consisting in 278 amino acids. At the nucleotide sequence level, BrNAC has 97% sequence similarity to Brassica napus NAC5-8, 92% to NAC5-7, 86% to NAC3 genes and 89% to Arabidopsis thaliana AFAF2. The secondary structure analysis revealed that BrNAC strongly resembled other NAC genes. BrNAC was more close to AFAF2 in its homologous evolutional tree. The result of RT-PCR indicated that the expression of BrNAC gene was induced by cold stress, mechanical damage and high salinity, but not NAA and ABA, and the transcript level of BrNAC gene was the highest in Brassica campestris ssp. chinensis stem and leave.SP1 gene from Populus tremula and ThpI gene from Choristoneura fumiferana were successively synthesized by PCR-based two-step DNA synthesis (PTDS). It is a simple, high fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. Compared to the previously published methods, the PTDS method is rapid (6 to 7 days) and suitable for synthesizing different species and different segments, long segments of DNA (5 to 6-kb) with high G+C contents, repetitive sequences, or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures.We analyzed the function of expression of SP1 and ThpI gene in Arabidopsis thaliana plants, through constructing the Agrobacterium tumefaciens GV3101 containing plant binary expression vector. The floral dip method was used to transfer them into A. thaliana. The results showed that the SP1 transgenic plants exhibited improved growth than wild type control plants after 45℃high temperature stress for 16 h. The chlorophyll and water contents and chlorophyll fluorescence were decreased much more in wild type than in transgenic plants; Moreover, the transgenic plants had higher proline contents and lower malondialdehyde contents after high temperature stress. After low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild plants. The transgenic plants had higher proline contents and higher SOD activity. Moreover, they had lower electrolyte leakage percentage and lower malondialdehyde contents after cold stress.