Cloning and expression analysis of CtbHLH transcription factor gene from Carthamus tinctorius and construction of plant expression vector / 中国中药杂志

To clone bHLH( basic helix-loop-helix) gene from Carthamus tinctorius,analyze the expression level in different plant tissues and construct the plant expression vector. The bHLH1 gene was cloned by RT-PCR techniques,and the protein characteristics were analyzed by bioinformatics,and phylogenetic tree was constructed. The expression of bHLH1 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR,and the plant expression vector p BASTA-bHLH1 was constructed. The obtained ORF sequence of bHLH1 gene was 897 bp,encoded a protein of 298 amino acids. Sequence alignment and phylogenetic tree analyses showed that C. tinctorius bHLH1 had a certain homology with other species of amino acids,and was the most similar to the amino acid sequence of tobacco. Real-time PCR results showed significant differences,CtbHLH1 gene in red flower petals in different tissues and different flowering period had remarkable difference in expression level,its high amount expressed in petals,flowers third day after blossom expressed the highest quantity,at the end of the flowering the expression quantity is low. In addition,it is expressed in the root,and the expression in the stem and leaves is extremely low. The bHLH1 gene of C. tinctorius is successfully cloned,and the expression is analyzed. The plant expression vector p BASTA-bHLH is constructed.

Cloning of ERF1 transcription factor from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods: A gene (CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35S promoter. Results: CtERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The pBASTA-CtERF1 plant expression vector was constructed successfully by molecular biology. Conclusion: A CtERF1 gene of C. tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector pBASTA-CtERF1 was constructed successfully.

Cloning of full-length cDNA of gene encoding dihydrodipicolinate synthase from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone the full-length cDNA sequence of DHDPS gene encoding the key enzyme DHDPS in lysing biosynthesis pathway of Carthamus tinctorius, and to construct the plant expression vector. Methods: The dihydrodipicolinate synthase (DHDPS) gene fragment was acquired according to the sequence of transcriptome in C. tinctorius, and the full-length cDNA sequence of CtDHDPS gene from C. tinctorius seeds was cloned by RT-PCR and RACE technologies. The pBasta-DHDPS plant expression vector was constructed using traditional molecular cloning and recombination technique. Results: Bioinformatics analysis showed that the full-length cDNA of CtDHDPS was 1309 bp, open reading frame was 954 bp, encoding a polypeptide of 317 amino acids, the theoretical isoelectric point of the coded protein was 5.93, and the molecular weight was about 34 750.79. The plant expression vector pBasta-DHDPS was successfully constructed by traditional molecular cloning and recombinant technique. Conclusion: The full-length sequence of CtDHDPS gene is obtained and the plant expression vector is successfully constructed, which lays a foundation for the further study on the mechanism of CtDHDPS in regulation of essential amino acid metabolism.

Cloning and expression analysis of CtbZIP20 transcription factor gene from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone bZIP20 (basic region/leucine zipper motif) gene from Carthamus tinctorius, analyze the expression level in different plant tissues, and construct the plant expression vector. Methods: The bZIP20 gene was cloned by RT-PCR techniques, and the protein characteristics were analyzed by bioinformatics, and phylogenetic tree was constructed. The expression of bZIP20 gene in different tissues and the roots after inoculated by Fusarium oxysporum were analyzed using real time-PCR, and the plant expression vector pBASTA-bZIP20 was constructed. Results: The ORF sequence of bZIP20 gene was 981 bp, encoded a protein of 326 amino acids (GenBank: KT692605). Sequence alignment and phylogenetic tree analyses showed that bZIP20 had 85.41% and 83.99% of consistency with bZIP of Sesamum indicum and Camellia assamica. Real-time PCR results showed significant differences, the highest expression level of bZIP20 gene was detected in flower, and was highest in the bud period, bZIP20 gene was significantly increased in root tissue inoculated with F. oxysporum. The plant expression vector pBASTA-bZIP20 was obtained. Conclusion: The bZIP20 gene of safflower is successfully cloned, and the expression is analyzed. The plant expression vector pBASTA-bZIP20 is constructed.

Cloning of Ginseng ? AS Gene and the Construction of Its Antisense Plant Expression Vector / 中国生物工程杂志

RT-PCR amplification of ginseng ?-amyrin synthase gene was successfully performed based on the total RNAs extracted from ginseng hairy roots induced by Agrobacterium rhizogenes A4 using a modified guanidine isothiocyanate-method. Sequence analysis of this gene revealed that its sequence was consistent with the sequence of a previously reported ginseng ?-amyrin synthase gene (GenBank No. AB009030). This gene was inserted into pMD-119T simple vector and transformed into E.coli DH5?. Furthermore antisense plant expression vector of this gene was constructed using the pBI121 vector, laying foundation for studies on antisense regulation of ginseng ?-amyrin synthase gene.

Cloning, Sequencing of Suaeda heteroptera kitag CMO cDNA and Construction of its Recombinant Plant Expression Vector / 中国生物工程杂志

Total RNA was extracted from leaf of Suaeda hetroptera kitag, then the CMO ( choline monooxygenase) cDNA was amplified using the reverse transcriptase polymerase chain reaction ( RT-PCR) method and cloned into pMD-T-simple vector. The positive clones from the Blue/White Screen were sequenced. After confirming its validity, the CMO gene fragment was cloned into pBI121 vector. Double enzyme restriction and PCR analysis indicated that the pBI121/CMO recombinant plasmid was successfully constructed.