Screening And Identification On The Binding Sequence Of SOC1 Promoter Interacted With The Flower Regulator FLC And SVP In Brassica Juncea

Brassica juncea is one of the extremely important domestic cruciferous vegetables, which is widely planted in our country. For now we only know a little about the details of molecular mechanism of mustard flowering regulation. In vernalization pathway and autonomous pathway, FLC plays an important regulatory role to suppress the blossom, while in gibberellins-induced pathway and photoperiod, SVP also can inhibit the plants blossom. SOC1 that located in the downstream of FLC SVP is able to integrate signals from the four regulation pathways of flowering, the expression of SOC1 is negatively regulated by FLC、SVP protein. FLC and SVP which belong to MADS transcription factors which can encode MICK type protein, the MICK-type protein can identify the specific CArG-box sequence.According to the result of previous experiment,our laboratory have proved that in Brassica juncea FLC and SVP protein can identify and combine to the SOC1 promoter fragment(a length of 782 bp DNA fragments upstream of the coding sequence). But the test did not screen the clear section on the SOC1 promoter that is specifically identified and combined by FLC and SVP protein. Our study is based on the result that there is an interaction between the FLC% SVP protein and the SOC1 promoter. We carried on a further experiment to screen the clear site on the SOC1 promoter which is specifically identified and combined by FLC and SVP protein, and we try to figure out whether the DNA-binding site have specificity or not. The result of our research could make contribute to better understanding of the expression and regulation of SOC1 in molecular mechanism of mustard flowering regulation.1 The fragmentation cloning of the SOC1 promoter and the action between the small fragmentation and FLC、SVP protein in Brassica junceaWe utilized the PCR to cut the SOCl promoter into three fragments, each of the fragments has a CArG-box-like(CC[A/T]6GG-like). Finally we named the three fragments:SOC1-1、SOC1-2 and SOC1-3. Then we respectively connected the three small fragments to the yeast vector plasmid pAbAi to build the yeast recombinant plasmid.Then we transited the recombinant plasmid into the yeast strain YIH Gold to recreate the recombinant yeast Y1H[pAbAi-SOC1△1-3]. Then we screened the lowest concentration of the Aureobasidin A (AbA) that inhibits the growth of Y1H[pAbAi-SOC1△1-3]is 100ng/ml、150ng/ml、100ng/ml.Then we put the recombinant plasmid pGADT7FLC、pGADT7SVP which can code the fusion protein FLC、SVP into the recombinant yeast YIH to get the recombinant yeast Y1H[SOC1△1-3+FLC] and Y1H[SOC1△1-3+SVP].We used the Yeast one-hybrid system to test whether the recombinant yeast Y1H[SOC1△1-3+FLC] and Y1H[SOC1△1-3+SVP] can grow normally on the medium SD/-Leu/AbA* which contain the corresponding background concentration of AbA. The result could proved that there is a interaction between SOC1△1-3 and FLC、SVP protein.2 The interaction between the SOC1 promoter type Ⅰ mutant and FLC、 SVP protein in Brassica junceaWe eliminated the CArG-box-like in SOC1△1-3 to get the SOC1 promoter type I mutant, we named them:Eliminate1、Eliminate2 and Eliminate3. Then we got the Y1H[pAbAi-[Eliminate △ 1-3], and we screened the lowest concentration of the Aureobasidin A (AbA) that inhibits the growth of Y1H[pAbAi-Eliminate△1-3] is 100ng/ml、150ng/ml、100ng/ml.We created the recombinant yeast Y1H[Eliminate△1-3+FLC] and Y1H[Eliminate △1-3+SVP] as well. Further We use the Yeast one-hybrid system to test whether the recombinant yeast Y1H[Eliminate △ 1-3+FLC] and Y1H[Eliminate△1-3+SVP] can not grow normally on the medium SD/-Leu/AbA* which contain the corresponding background concentration of AbA. The result could prove that there is no interaction between the SOC1 promoter type Ⅰ mutant (Eliminate△1-3) and FLC、SVP protein. So we could make a conclusion that the CArG-box-like on the SOC1 promoter is the clear site which is specifically identified and combined by FLC and SVP protein.3 The interaction between the SOC1 promoter type Ⅱ mutant and FLC、 SVP protein in Brassica junceawe exchange the base sequence of A and T in CArG-box-like. So we got the SOC1 promoter type Ⅱ mutant. We named them:Exchange1、Exchange2 and Exchange3. In the same way, we screened the lowest concentration of the Aureobasidin A (AbA) that inhibits the growth of YlH[pAbAi-Eliminate△1-3] is 100ng/ml、 200ng/ml、200ng/ml.Then we created the recombinant yeast YlH[Exchange △ 1-3+FLC] and YlH[Exchange△1-3+SVP]. Further We use the Yeast one-hybrid system to test whether the recombinant yeast YlH[Exchange△1-3+FLC] and YlH[Exchange△ 1-3+SVP] can not grow normally on the medium SD/-Leu/AbA* which contain the corresponding background concentration of AbA. The result could prove that there is no interaction between the SOC1 promoter type Ⅱ mutant (Exchange△1-3) and FLC、SVP protein, which explained that the FLC、SVP protein would recognize the specific CArG-box sequence, and if the CArG-box sequence has been slightly different, the FLC、SVP protein can not be specifically identified and combined to the sequence any more.