灌木柳SlSIP基因的克隆和功能验证
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摘要
【目的】明确灌木柳碱性α-半乳糖苷酶基因(Sl SIP)的结构特征和该基因在耐盐方面的生物学功能,为碱性α-半乳糖苷酶基因新的功能拓宽提供理论基础。【方法】以灌木柳苏柳‘2345’(Salix jiangsuensis‘2345’)盐胁迫48 h后的c DNA为模板,克隆了苏柳碱性α-半乳糖苷酶基因Sl SIP。运用生物信息学方法分析其蛋白质结构、性质和亲缘关系,利用农杆菌介导法转入拟南芥中,通过实时荧光定量qRT-PCR方法鉴定转基因阳性植株的表达特性和盐胁迫条件下的生长状态,从而验证转基因植株的耐盐功能。【结果】该基因编码776个氨基酸,分子质量为84.88 ku,理论等电点为5.85,不稳定系数为35.74,亲水性平均系数-0.154,定位于内质网膜上。SlSIP的保守结构域为WFGWCTW和IDDGWQ,催化活性位点为V。共得到11个T3代阳性转基因株系,qRT-PCR结果显示Sl SIP在3个拟南芥阳性转基因株系中表达量最高,分别提高9、28、29倍。转基因株系在含85 mmol/L Na Cl培养基上种子萌发率高于非转基因植株,且生长状态良好,但非转基因植株的叶绿素含量高于转基因株系。【结论】Sl SIP基因属于GH27家族,不仅提高了种子萌发过程中的耐盐性,也参与了叶片发育和衰老的途径。
【Objective】The objectives of this study were to elucidate the structural characteristics and biological functions of the Sl SIP gene from shrub Salix and to provide a theoretical basis for the new function of alkaline alpha-galactosidase gene.【Method】An alkaline α-galactosidase gene encoding Sl SIP was cloned from the c DNA template of Salix jiangsuensis'2345'after salt stress for 48 h. A bioinformatics method was used to analyze the protein structure and relationships. The expression level and function of Sl SIP were conducted through agrobacterium-mediated transformation into Arabidopsis thaliana and real-time PCR. 【Result】This gene encoded 776 amino acids with a molecular weight of 84.88 ku,a theoretical isoelectric point of 5.85,an unstable coefficient of 35.74,and the average hydrophilic coefficient of-0.154. It was located on the endoplasmic reticulum. The conserved domains of Sl SIP were WFGWCTW and IDDGWQ with the catalytic active site V. In total,11 positive T3 generation transgenic homozygotes were obtained. qRT-PCR showed that the expression level of Sl SIP increased by 9,28,29 times in 3 Arabidopsis transgenic lines. The seed germination rate of the transgenic lines was higher than that of the wild type on 85 mmol/L Na Cl medium,and the growth state was well,but the chlorophyll content of the wild-type plants was higher than that of the transgenic lines. 【Conclusion】Sl SIP belongs to the GH27 gene family. This indicated that the Sl SIP gene not only increased the salt tolerance of seed germination,but also participated in leaf growth and senescence. This is the first study identifying the novel function of an alkaline α-galactosidase gene.
【Objective】The objectives of this study were to elucidate the structural characteristics and biological functions of the Sl SIP gene from shrub Salix and to provide a theoretical basis for the new function of alkaline alpha-galactosidase gene.【Method】An alkaline α-galactosidase gene encoding Sl SIP was cloned from the c DNA template of Salix jiangsuensis'2345'after salt stress for 48 h. A bioinformatics method was used to analyze the protein structure and relationships. The expression level and function of Sl SIP were conducted through agrobacterium-mediated transformation into Arabidopsis thaliana and real-time PCR. 【Result】This gene encoded 776 amino acids with a molecular weight of 84.88 ku,a theoretical isoelectric point of 5.85,an unstable coefficient of 35.74,and the average hydrophilic coefficient of-0.154. It was located on the endoplasmic reticulum. The conserved domains of Sl SIP were WFGWCTW and IDDGWQ with the catalytic active site V. In total,11 positive T3 generation transgenic homozygotes were obtained. qRT-PCR showed that the expression level of Sl SIP increased by 9,28,29 times in 3 Arabidopsis transgenic lines. The seed germination rate of the transgenic lines was higher than that of the wild type on 85 mmol/L Na Cl medium,and the growth state was well,but the chlorophyll content of the wild-type plants was higher than that of the transgenic lines. 【Conclusion】Sl SIP belongs to the GH27 gene family. This indicated that the Sl SIP gene not only increased the salt tolerance of seed germination,but also participated in leaf growth and senescence. This is the first study identifying the novel function of an alkaline α-galactosidase gene.
引文
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[12]徐晓锋.黄瓜α-半乳糖苷酶基因克隆及表达分析[D].扬州:扬州大学,2006.XU X F. Cloning and expression analysis ofα-galactosidase in Cucumber(Cucumis sativus L.)[D]. Yangzhou:Yangzhou University,2006.
[13]MATHEW C D,BALASUBRAMANIAM K. Mechanism of action ofα-galactosidase[J]. Phytochemistry,1987,26:1299-1300.DOI:10.1016/S0031-9422.10028.1798-7.
[14]CHIEN S F,CHEN S H,CHIEN M Y. Cloning,expression,and characterization of riceα-galactosidase[J]. Plant Molecular Biology Reporter,2008,26(3):213-224. DOI:10. 1007/s11105-008-0035-6.
[15]PENNYCOOKE J C,VEPACHEDU R,STUSHNOFF C,et al.Expression of anα-galactosidase gene in Petunia is upregulated during low-temperature deacclimation[J]. Journal of the American Society for Horticultural Science,2004,129(4):491-496.
[16] SAMIA D,ANIS B A,MAHOUD G,et al. A grapevineinducible gene Vv-α-gal/SIP confers calt and desiccation tolerance in Escherichia coli and tobacco at germinative stage[J]. Biochemical Genetics,2017,56(1):1-15. DOI:10. 1007/s10528-017-9831-8.
[2]FOLMER F,MAHER A H,RENE J L,et al. Crystal structure ofα-galactosidase from lactobacillus acidophilus NCFM:Insight into tetramer formation and substrate binding[J]. Journal of Mobecular Biology,2011,412(3):466-480. DOI:10.1016/j.jmb.2011.07.057.
[3]郝桂娟,张凯,王学智,等.α-半乳糖苷酶的研究进展[J].中国畜牧兽医,2013,40(3):149-154. DOI:10.3969/j.issn.1671-7236.2013.03.034.HAO G J,ZHANG K,WANG X Z,et al. Research progress onα-galactosidase[J]. China Animal Husbandry&Veterinary Medicine,2013,40(3):149-154.
[4]YU J H,GUO T T,XUE R G,et al. A protease-resistantα-galactosidase from Pleurotus citrinopileatus with broad substrate specificity and good hydrolytic activity on raffinose family oligosaccharides[J]. Process Biochem,2016,51(4):491-499. DOI:10.1016/j.procbio.2016.01.010.
[5] GAUDREAULT P R,WEBB J A. Alkalineα-galactosidase in leaves of Cucurbita pepo[J]. Plant Science Letters,1982,24(3):281-288. DOI:10.1016/0304-4211(82)90023-2.
[6]GAO Z,SCHAFFER A A. A novel alkalineα-galactosidase from melon fruit with a substrate preference for raffinose[J]. Plant Physiology,1999,119(3):979-988. DOI:10.1104/pp.119.3.979.
[7]KANG N J,SHON Y G,RHO I R,et al. Changes in carbohydrates accumulation and metabolic enzyme activity during fruit development and maturation in Cucurbita maxima and Cucurbita pepo[J]. Changes,2002,43(5):571-574.
[8]HARA M,TOKUNAGA K,KUBOI T. Isolation of a drought-responsive alkalineα-galactosidase gene from New Zealand spinach[J]. Plant Biotechnology,2008,25(5):497-501. DOI:10.5511/plantbiotechnology.25.497.
[9]YANG D X,TIAN G T,DU F,et al. A fungal alpha-galactosidase from Pseudobalsamia microspora capable of degrading raffinose family oligosaccharide[J]. Applied Biochemistry and Biotechnology,2015,176(8):2157-2169. DOI:I10. 1007/s12010-015-1705-0.
[10] MITTAL Y,SHARMA C B. Development ofα-galactosidase isoenzymes in chickpea seeds[J]. Plant Sci,1991,77,185-190.
[11] FERTADO J A,BANIK M,BEWLEY J D. The cloning and characterizaion ofα-galactosidase present during and following germination of tomato(Lycoperisicon esculentum Mill.)seed[J].Journal of Experimental Botany,2001,52,1239-1249.
[12]徐晓锋.黄瓜α-半乳糖苷酶基因克隆及表达分析[D].扬州:扬州大学,2006.XU X F. Cloning and expression analysis ofα-galactosidase in Cucumber(Cucumis sativus L.)[D]. Yangzhou:Yangzhou University,2006.
[13]MATHEW C D,BALASUBRAMANIAM K. Mechanism of action ofα-galactosidase[J]. Phytochemistry,1987,26:1299-1300.DOI:10.1016/S0031-9422.10028.1798-7.
[14]CHIEN S F,CHEN S H,CHIEN M Y. Cloning,expression,and characterization of riceα-galactosidase[J]. Plant Molecular Biology Reporter,2008,26(3):213-224. DOI:10. 1007/s11105-008-0035-6.
[15]PENNYCOOKE J C,VEPACHEDU R,STUSHNOFF C,et al.Expression of anα-galactosidase gene in Petunia is upregulated during low-temperature deacclimation[J]. Journal of the American Society for Horticultural Science,2004,129(4):491-496.
[16] SAMIA D,ANIS B A,MAHOUD G,et al. A grapevineinducible gene Vv-α-gal/SIP confers calt and desiccation tolerance in Escherichia coli and tobacco at germinative stage[J]. Biochemical Genetics,2017,56(1):1-15. DOI:10. 1007/s10528-017-9831-8.