Cloning And Functional Analysis Of BnTT8 Transcription Factor In Brassica Napus

Previous studies have revealed that Arabidopsis AtTT8,a member of the bHLH(basic helix-loop-helix)transcription factors family,not only involves in the biosynthesis of anthocyanins and procyanidins,but also affects seed development and the accumulation of seed storage reserves in Arabidopsis.To investigate whether the homologous gene BnTT8 from Brassica napus has similar functions with AtTT8,we cloned the full-length coding domain sequence(CDS)of BnTT8 from QINYOU Seven,a cultivar of Brassica napus,carried out bioinformatics analysis and tobacco leaf subcellular localization,and analyzed the spatio-temporal expression pattern of BnTT8 in different Brassica napus tissues.In addition,we transformed the recombinant vector 35S:BnTT8 to the Arabidopsis mutant tt8-4 and successfully obtained the T3 homozygous transgenic plants of tt8-4 35S:BnTT8.Moreover,we analyzed the effects of BnTT8 on multiple biological processes,such as anthocyanin and procyanidin biosynthesis,seed storage reserve accumulation,as well as the responses to abiotic stresses.Furthermore,we transformed BnTT8-RNAi(RNA interference)into an inbred line L111 of Brassica napus and obtained transgenic plants.The main results for the experiment were listed as follows:1.The BnTT8 gene was cloned from Brassica napus cultivar QINYOU Seven,and its protein sequence was aligned with TT8 protein sequences of Brassica napus and Arabidopsis in the NCBI database.The results showed that the similarity of the protein sequence of BnTT8 and AtTT8 was very high and the protein functional domain was highly conserved,which indicates that BnTT8 and AtTT8 may have similar physiological and biochemical functions.2.The tobacco leaf subcellular localization indicated that BnTT8 was located in the nucleus,and it can be speculated that BnTT8 functions as a transcription factor.RT-qPCR results showed that BnTT8 was expressed in different tissues of Brassica napus and exhibited highest expression level in the developing seeds.This suggested that the transcription factor may play roles in multiple biological processes(especially seed development)of plant growth and development.3.The constructed recombinant vector 35S:BnTT8 was transformed into Arabidopsis mutant tt8-4 plants.The homozygous plants obtained were identified by PCR.Both at the level of DNA and RNA,we confirmed that we have successfully obtained transgenic plants of tt8-4 35S: BnTT8.4.The seed phenotypes of the wild-type,tt8-4 and homozygous transgenic plant tt8-435S:BnTT8 were analyzed.The results showed that the overexpression of BnTT8 in the background of the tt8-4 mutant can restore the seed coat color of the mutant and the phenotypes of the higher seed fatty acids and low storage protein to the level of wild type.5.Under the abiotic stresses of 100 mM NaCl and 3%(w/v)glucose,the transgenic plant of tt8-4 35S:BnTT8 can restore the mutant tt8-4 phenotype to the wild type level,including delayed seed germination and seedling growthd.This result indicated that the functions of BnTT8 was similar to AtTT8 and was involved in the regulation of seed germination and seedling establishment under environmental stresses.6.The analysis of stress-response genes showed that without any stresses,except for AtABA1,AtNCED3,and AtRAB18,the expression levels of other important genes have no significantly differences between wild type and the mutant.Under both the high salt and glucose stresses,the transcriptional expression of AtABI2,AtCBL1,AtCBL9,AtNCED3,AtRD29 A,AtRD22,and AtKINI was significantly different between wild type and the mutant.The expression levels could be restored to the levels of wild type for the above mentioned genes in the transgenic plants tt8-4 35S:BnTT8.Therefore,it can be supposed that BnTT8 might be involved in the control of the expression of stress-response genes.7.Preliminary identification results showed that we successfully obtained Brassica napus transgenic plants of BnTT8-RNAi.In this study,BnTT8 functions were verified by transforming BnTT8 into tt8-4 mutant.The comparisons of similarities and differences of BnTT8 and AtTT8 in multiple biological processes during plant growth and development,which not only lets us know more about the functions of TT8 from Arabidopsis and Brassica napus,but also provides a key gene for Brassica napus breeding to improve seed yield and quality and other important agronomic traits.