The Response To Drought Stress And The Relationship With ABA Of CAM Gene Of Sinapsis Alba L.

In order to reveal the molecular mechanism of drought resistance of Sinapsis alba L.(Sinapsis alba L.). This study clones Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L., and explores the response to drought stress and the expression pattern under ABA treatments of those genes. In this study, Sinapsis alba L. is used as examination material. We use hydroponics to culture Sinapsis alba L. And PEG6000 is used as drought stress to treat Sinapsis alba L. seedlings, which is also treated with exogenous ABA. The main results are as followings:1) Based on the CDS sequences witch have been reported in NCBI of Ca M1, Ca M2 and Ca M3 gene of Arabidopsis thaliana, We design their primers with Primer5. The Ca M1, Ca M2 and Ca M3 gene fragments are got from Sinapsis alba L. leaves through homologous gene cloning. The effective length of these fragments are 278 bp, 302 bp and 273 bp seperately. Specific primers are designed according to the above Ca M gene sequences of Sinapsis alba L. The full-length of c DNA sequences are obtained by using the technology of RACE(Ca M1 1128 bp, Ca M2 762 bp and Ca M3 1095 bp). And the sequences of CDs are found out through BLAST(Ca M1 453 bp, Ca M2 453 bp and Ca M3 450 bp). By analysising homology of the three genes with blast and DNAMAN software, the result we get shows that the homology of Ca M1, Ca M2 and Ca M3 genes of Sinapsis alba L. corresponded with other species are very high. So we speculate that the three genes are Ca M gene of Sinapsis alba L. And through the analysis of the DNA sequence characteristics, two level structures of nucleic acid, two level structures of protein, three level structures of protein and dihedral angles of protein of the three Ca M genes. The Ca M genes which is got from cloning and is confirmed as Ca M1,Ca M2 and Ca M3 gene.2) Sinapsis alba L. seedlings were treated with 25% PEG6000 by 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h and 36 h to simulate Drought stress. Each treatment group has three biological replicates. Samples are got after the end of those treatments(The Sinapsis alba L. witch is cultured in normal surroundings is a control group. The sampling time is same to the treatment groups). Meanwhile real-time fluorescence quantitative PCR is used to test the treatment groups content of Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L.. The results are as follows: The transcription and expression of three genes have the same trend; they show the law that the expression is increasing at first and then decreasing; and the highest expression appears at 8 h of the processing time. It is to say Sinapsis alba L. seedlings is treated with short time of drought stress and Sinapsis alba L. responses to stress by increasing the expression of Ca M gene and gradually adapts to the stress until the genes’ expression recover.3) In order to study the relationship between Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L. and ABA,We use exogenous 100 μmol·L-1 ABA to treat Sinapsis alba L. seedlings, which are also treated with 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h and 36 h. Each treatment group has three biological replicates. Samples are got after the end of those treatments(A group witch is cultured in normal surroundings is a control group, whose sampling time is same to the treatment groups).The results of real time fluorescent quantitative expression of Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L. are similar to under drought stress groups. At the early stage of the treatment of exogenous ABA, Ca M genes expression of Sinapsis alba L. leaves increases; with the treatment time extensing, the Ca M gene expression decreases,which proves that Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L. response to exogenous ABA.4) We use different concentration of fluridone(FLU) solution which is ABA inhibitor to treat Sinapsis alba L. seeds. The FLU solution can make the Ca M genes expression of Sinapsis alba L. inhibite. We select FLU solution of 0.01 μmol·L-1 to treat Sinapsis alba L. seeds. After cultivating Sinapsis alba L. seedlings, we add exogenous ABA, the expression of Ca M genes are strengthed compared with the normal culture Sinapsis alba L. seedlings. And when we block the synthesis of endogenous ABA, the expression of Ca M genes decreases. It decilines that the expression patterns of Ca M1, Ca M2 and Ca M3 gene of Sinapsis alba L. belong to the ABA dependent. By additing exogenous ABA to normal cultured seedlings under drought stress, the expression of the three Ca M genes of Sinapsis alba L. are higher than those are only treated with drought stress. And the expression of Ca M genes of the above three kinds of treatments are higher than those of the control group. It is showed that the drought stress and ABA play a synergistic role in Ca M genes expression, which jointly promote the genes responding to stress.