Cloning And Functional Analysis Of Glyphosate-induced Highly Expression Gene And Its Promoter

Glyphosate is a widely used, broad-spectrum, inner-absorption conducting herbicide. Since it is also harmful to crops, areas and time of usage are extremely limited. In 2004, the estimated global area of approved GM crops was 81.0 million hectares and herbicide-resistant transgenic plants occupied 72%. With the large-scale planting of glyphosate-resistant crops all over the world, applications of glyphosate become increasingly broader.Currently, constitutive promoters were used in almost all reported glyphosate-resistant transgenic plants. However, due to their constitutive expression pattern, extraneous genes can express in all tissues of plants at any development stage, which would lead to great waste of both materials and energy and increase negative influences in metabolism for host plants. Therefore, researches on specific promoters, ideal alternatives to constitutive promoters, have become a focus on plant genetic engineering. In this study, a specific ghphosate-induced promoter from cotton was isolated and identified, which can drive the extraneous glyphosate-resistant gene to just highly express after spraying glyphosate.After screening 40 pairs of primer by DDRT-PCR, we obtained 65 EST (31 upstream- regulate fragments, 8 downstream-regulate fragments from soybean and 25 upstream- regulate fragments, 1 downstream-regulate fragment from cotton), which were expressed obviously different after glyphosate-induced. The Blast result revealed that theses EST sequences have at least 82% homology with sequences induced by other abiotic stress factors ( NaCl, Chill etc.). The most dramatically high-level expression gene was screened by RT-PCR. It was named ag2 gene amplified by T7T12AG and M13RP2 primers from cotton. Northern blot analysis indicated that ag2 gene was glyphosate-induced gene.The full length of ag2 cDNA sequences (763bp) was isolated via RACE, and the corresponding fragment (856bp) in cotton genome was also amplified. The encoding region of ag2 gene contains 456bp and encodes 152 amino acid residues, including 2 exons and 1 intron. Sequence analysis demonstrated that ag2 would be interrelated with resisting stress effects. As a result of no report about functions of ag2 gene presented, we proposed that it would be a novel gene which function is unknown. Thereafter, we constructed plant expression vectors pBIag2A, pBIag2S, and transferred ag2 into cotton variety Y18 via pollen tube and 1.73Kg seeds were obtained. Thirty-three positive strains carrying pBIag2S and 18 positive strains carrying pBIag2A were detected by PCR. At the same time, 29 positive transgenic tobaccos were also obtained. Results of stress-resisted tests showed ag2 gene has resistant effects to SA, Fusarirum wilt and NaCI to some extent in T1 generation transgenic tobaccos. It indicated that the product expressed by ag2 gene would be a member of signal pathway or final product in inducing reaction. In sum, the product highly expressed by ag2 gene would be related to plant stress-resistance process or could abate the stress directly.A promoter (2063bp) of ag2 was forwarded from its initiator to 5' by thrice chromosome walking using hemispecific PCR and Cassette ligation PCR. This promoter is not registered in GenBank. Based...