[1]周 洁,黄 婧,邢 玮,等.灌木柳SlSIP基因的克隆和功能验证[J].南京林业大学学报(自然科学版),2019,43(01):039-44.
 ZHOU Jie,HUANG Jing,XING Wei,et al.Cloning and functional characterization of SlSIP from shrub Salix[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(01):039-44.
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灌木柳SlSIP基因的克隆和功能验证
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
43
期数:
2019年01期
页码:
039-44
栏目:
研究论文
出版日期:
2019-01-28

文章信息/Info

Title:
Cloning and functional characterization of SlSIP from shrub Salix
文章编号:
1000-2006(2019)01-0039-06
作者:
周 洁黄 婧邢 玮王保松何旭东教忠意
江苏省林业科学研究院, 江苏 南京 211153
Author(s):
ZHOU Jie HUANG JingXING Wei WANG Baosong HE Xudong JIAO Zhongyi
Jiangsu Academy of Forestry, Nanjing 211153, China
关键词:
苏柳‘2345’ 碱性α-半乳糖苷酶 耐盐 转基因 拟南芥 功能验证
Keywords:
Salix jiangsusuensis ‘2345’ alkaline α-galactosidase salt tolerance transgenic Arabidopsis thaliana functional annotation
分类号:
Q786
文献标志码:
A
摘要:
【目的】明确灌木柳碱性α-半乳糖苷酶基因(SlSIP)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cDNA为模板,克隆了苏柳碱性α-半乳糖苷酶基因SlSIP。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SlSIP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 NaCl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】SlSIP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。
Abstract:
【Objective】 The objectives of this study were to elucidate the structural characteristics and biological functions of the SlSIP 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 SlSIP was cloned from the cDNA 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 SlSIP 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 SlSIP 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 SlSIP 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 NaCl 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】 SlSIP belongs to the GH27 gene family. This indicated that the SlSIP 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.

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备注/Memo

备注/Memo:
收稿日期:2018-08-03 修回日期:2018-09-19基金项目:国家自然科学基金项目(31400572); 江苏省自然科学基金项目(BK20141039)。 第一作者:周洁(zjwin718@126.com),高级工程师,ORCID(0000-0001-8214-8479)。引文格式:周洁,黄婧,邢伟,等. 灌木柳SlSIP基因的克隆和功能验证[J]. 南京林业大学学报(自然科学版),2019,43(1):39-44.
更新日期/Last Update: 2019-01-28