Cloning And Analyzing AGL24 Promoter And The Genetic Transformation Of Its Trans-acting Element SOC1 In Brassica Juncea Coss.

Brassica juncea is a famous kind of Brassica vegetable crops. In science study, it is a meaningful work to study characters and regulation mechanisms of bolting and flowering time in Brassica juncea and it will provide valuable information for future research on cultivation and breeding. In recent years, more than 180 genes related to flowering genetic regulatory network have been identified in the model plant of Arabidopsis thaliana. And they have been classified into 6 flowering regulation pathways as follows: photoperiod pathway, vernalization pathway, ambient temperature,age pathway, autonomous pathway and gibberellin(GA) pathway. These flowering signals in different pathways can be integrated into floral integrators which form an integrative network and finally regulate the bolting and flowering time.. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1) and AGAMOUS-LIKE24(AGL24)are two vital flowering signal integrators among them. Previous studies have showed that AGL24 and SOC1 act as cross-talk loop in Arabidopsis thaliana. SOC1 and AGL24 are able to regulate each other at the mRNA levels, that is, mutually up-regulate the two genes to high expressions and finally promote flowering. However, it is still unknown whether Brassica juncea SOC1 and AGL24 have the same mechanisms as those of Arabidopsis and how they regulate each other in flowering time.To clarify the interaction mechanism between SOC1 protein and AGL24 gene in Brassica juncea, this study focused on checking whether the SOC1 protein can be used as a transcription factor to interact with AGL24 promoter, and then to up-regulate AGL24 expression. First, according to the known gene sequence of AGL24, wesuccessfully cloned the promoter of AGL24 gene using the genome walking method.Subsequently, we broke the AGL24 promoter into 3 fragments, along with SOC1, and then built up yeast one-hybrid recombinant expression vectors. By the means of yeast one-hybrid system, the interactions and function area between them are analyzed.Finally, the anti and sense of SOC1 transgenic mustard system are also obtained. So it makes the foundation for further studying of the regulatory mechanisms in flowering time of Brassica juncea by regulating interactions between SOC1 protein and AGL24 promoter. The results are detailed as below.1. Cloning and bioinformatics analysis of AGL24 promoterWe extracted genome DNA from the leaves of Brassica juncea and designed the specific primers in according to the known AGL24 sequence. Using Genome Walking method, we successfully obtained 3020 bp sequence of 5’ end upstream promoter regions of AGL24 gene. Sequence analyzed by NCBI, we preliminarily predicateed it’s the 5’end promoter. Bioinformatics analysis of cis-acting elements indicated that it contained193 regulate elements such core promoter elements as TATA-box, CAAT-box and so on.We had analyzed and found that there are 16 similar CArG-boxs in the promoter which are probably bound by MADS-box transcription factors and play a vital role in mediating flowering time. It indicates that AGL24 may be regulated by a lot of MADS-box transcription factors in flowering, which is just worthy of the name of floral integrator. However, it is still unknown and need further study to uncover which MADS-box transcription factors are probably interacted with AGL24 promoter..2. The yeast one-hybrid experiment between AGL24 promoter truncated forms and SOC1 proteinTo further determine the interaction between SOC1 protein and AGL24 promoter in mustard, we first analyzed the AGL24 promoter, then truncated the full of AGL24 promoter into 3 fragments termed as AGL24 A, AGL24 B and AGL24 C, respectively.Subsequently, we constructed the appropriate yeast one-hybrid bait expression vectors termed as pAbAi-AGL24 A, pAbAi-AGL24 B, pAbAi-AGL24 C.respectively. We also constructed yeast one-hybrid prey vector named as pGADT7-SOC1. The above yeast recombinant plasmids were confirmed via PCR technique, sequencing and double digestion with restriction enzymes.Using yeast transformation system user manual, we integrated the linearizedpAbAi-AGL24 A, pAbAi-AGL24 B, pAbAi-AGL24 C plasmids into competent Y1 H Glod yeast cells and got yeast bait transformants of Y1H[pAbAi-AGL24A],Y1H[pAbAi-AGL24B] and Y1H[pAbAi-AGL24C]. The above yeast transformants failed to grow on the SD /-Ura plates supplemented with 350 ng/ml AbA, suggesting that 350 ng/ml AbA is an optimum concentration of inhibitory background and is suitable for screening and testing interactions of AGL24 promoter and other proteins.Subsequently, protein plamid of pGADT7-SOC1 was respectively transformed into Y1 H [pAbAi-AGL24A], Y1 H [pAbAi-AGL24B] and Y1 H [pAbAi-AGL24C], and then formed co-transformed yeast strains termed as Y1H[SOC1+AGL24A],Y1H[SOC1+AGL24B], Y1H[SOC1+AGL24C], respectively. After PCR checked, the above co-transformed strains were respectively coated on SD/-Leu+AbA350 media and cultured at 30 ℃ for 5-7 days. Meanwhile, Y1H[p53-AbAi+ pGADT7-53]and Y1H[pAbAi-AGL24A+ pGADT7] were taken as positive and negative controls,respectively. The results showed that only Y1H[SOC1+AGL24B] grew normally while both Y1H[SOC1+AGL24A] and Y1H[SOC1+AGL24C] failed to grow on the plates. It suggested that SOC1 protein of Brassica juncea could interact with truncated form of AGL24 B which was located in the middle zone of AGL24 promoter.3. Construction of anti and sense of SOC1 expression vectors and the genetic transformationBased on the sequence of SOC1 gene, we designed primers with BamH I on 5’ terminals and then sub-cloned SOC1 gene from the cDNA of Brassica juncea. Subsequently, we insert SOC1 into pBI121 vector forwardly and reversely. Using PCR methods to screen the positive recombinant plasmids, we obtaind anti and sense plamids termed as pBI121-SOC1 and pBI121-soc1 respcetively. Empty vector pBI121 was taken as a negative control, pBI121-SOC1 and pBI121-soc1 were respectively transformed into mustard hypocotyls by means of Agrobacterium-mediated genetic transformation. After a series of screening resitant plants on media with kanamycin, testing interest bands of PCR products on gels and staining tissues and organs to test the expression of GUS report gene, 14 sense and 8 anti SOC1 plants were obtained.