以耐盐碱野生大豆(Glycine soja L.G07256)为材料, 采用同源克隆方法和RT-PCR技术获得一个TIFY 类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明, 与其他物种相比, GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高, 达到56%; 序列分析表明GsTIFY11b蛋白除具有 TIFY保守结构域外, 还具有一个N端保守结构域和一个C端保守的Jas结构域; 实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达; 将GsTIFY11b转化拟南芥来验证其耐盐碱功能, 获得两个转基因纯合体株系, 盐碱胁迫分析结果表明, GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性, 并且与野生型相比, 转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明, GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明, GsTIFY11b定位于细胞核中。上述结果表明, 该基因在细胞核中起着转录调节子的作用, 可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。
Using homologous cloning and RT-PCR technology, we isolated a novel TIFY family gene, GsTIFY11b, from Glycine soja L. G07256, a species that is tolerant to saline and alkaline environments. Phylogenetic analysis indicated that GsTIFY11b was closely related to AtTIFY11a with 56% similarity in amino acid identity. Protein sequence analysis showed that GsTIFY11b protein also had conserved TIFY domain, N-terminal domain, and a C-terminal Jas motif. Quantitative realtime PCR analysis indicated that the expression of GsTIFY11b was induced by both saline and alkaline stresses. Two homozygous GsTIFY11b over-expressing transgenic Arabidopsis lines were obtained. Phenotypic analysis of the transgenic and wild-type Arabidopsis indicated that over-expressing GsTIFY11b in Arabidopsis did not enhance plant tolerance to saline and alkaline stresses, whereas it showed an increased sensitivity to saline stress during seed germination and seedling development. Expression analysis of saline stress response marker genes in transgenic and wild-type plants under stress condition indicated that GsTIFY11b regulated the expression of RD29B, KIN1, and DREB. The transient expression of a GsTIFY11b-GFP fusion protein in onion epidermal cells showed that GsTIFY11b was localized to the nucleus, suggesting a role as a transcriptional regulator in the saline stress response pathway.
[1] 朱丽萍, 于壮, 邹翠霞, 李秋莉. 植物逆境相关启动子及功能. 遗传, 2010, 32(3): 229-234.
[2] Vanholme B, Grunewald W, Bateman A, Kohchi T, Gheysen G. The tify family previously known as ZIM. Trends Plant Sci, 2007, 12(6): 239-244.
[3] Thines B, Katsir L, Melotto M, Niu YJ, Mandaokar A, Liu GH, Nomura K, He SY, Howe GA, Browse J. JAZ repres-sor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature, 2007, 448(7154): 661-665.
[4] Yan YX, Stolz S, Chételat A, Reymond P, Pagni M, Dubugnon L, Farmer E E. A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell, 2007, 19(8): 2470-2483.
[5] Ye HY, Du H, Tang N, Li XH, Xiong LZ. Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice. Plant Mol Biol, 2009, 71(3): 291-305.
[6] Ge Y, Li Y, Zhu YM, Bai X, Lv D-K, Guo DJ, Ji W, Cai H. Global transcriptome profiling of wild soybean (Glycine soja) roots under Na-HCO3 treatment. BMC Plant Biol, 2010, 10: 153.
[7] Willems E, Leyns L, Vandesompele J. Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem, 2008, 379(1): 127-129.
[8] Hussam HN, Bjarne GH, Morten HH, Jacob KJ, Barbara AH. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res, 2006, 34(18): e122.
[9] Clough SJ, Bent AF. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16(6): 735-743.
[10] Arnon D I. Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta vulgaris. Plant Physiol, 1949, 24(1): 1-15.
[11] Troll W, Lindsley J. A photometric method for the deter-mination of proline. J Biol Chem, 1955, 215(2): 655-660.
[12] Hagio T. Optimising the particle bombardment method for efficient genetic transformation. Jpn Agric Res Q, 1998, 32: 239-247.
[13] Staswick PE. JAZing up jasmonate signaling. Trends Plant Sci, 2008, 13(2): 66-71.
[14] Chini A, Fonseca S, Fernández G, Adie B, Chico JM, Lorenzo O, García-Casado G, López-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R. The JAZ family of repressors is the missing link in jasmonate signalling. Nature, 2007, 448(7154): 666-671.
[15] 周晓馥, 王兴智. 植物耐盐相关基因: SOS基因家族研究进展. 遗传, 2002, 24(2): 190-192.
[16] Wang H, Datla R, Georges F, Loewen M, Cutler AJ. Pro-moters from kin1 and cor6.6, two homologous Arabidopsis thaliana genes: transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration. Plant Mol Biol, 1995, 28(4): 605-617.
[17] Yamaguchi-Shinozaki K, Shinozaki K. Arabidopsis DNA encoding two desiccation-responsive rd29 genes. Plant Physiol, 1993, 101(3): 1119-1120.
[18] 王平荣, 邓晓建, 高晓玲, 陈静, 万佳, 姜华, 徐正君. DREB转录因子研究进展. 遗传, 2006, 28(3): 369-374.
[19] 葛瑛, 朱延明, 吕德康, 董婷婷, 王维世, 谭上进, 刘彩虹, 邹平. 野生大豆碱胁迫反应的研究. 草业科学, 2009, 26(2): 47-52.
[20] 王希, 李勇, 朱延明, 柏锡, 才华, 纪巍. 野生大豆胁迫应答膜联蛋白基因的克隆及胁迫耐性分析. 作物学报, 2010, 36(10): 1666-1673.
[21] Chung H S, Howe G A. A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis. Plant Cell, 2009, 21(1): 131-145.
