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林业科学 ›› 2015, Vol. 51 ›› Issue (4): 60-70.doi: 10.11707/j.1001-7488.20150408

• 论文与研究报告 • 上一篇    下一篇

桑树MLX56基因家族特性、克隆及表达分析

韩淑梅, 李军, 吕蕊花, 王晓红, 刘长英, 赵爱春, 鲁成, 余茂德   

  1. 西南大学生物技术学院 重庆 400715
  • 收稿日期:2014-06-27 修回日期:2014-10-12 出版日期:2015-04-25 发布日期:2015-05-20
  • 通讯作者: 余茂德
  • 基金资助:

    国家农业部公益性行业(农业)科研专项"蚕桑资源高值化加工利用技术及设备"(201403064); 国家现代农业产业技术体系建设专项(CARS-22)。

Characteristics, Cloning and Expression of the MLX56 Gene Family in Mulberry

Han Shumei, Li Jun, Lü Ruihua, Wang Xiaohong, Liu Changying, Zhao Aichun, Lu Cheng, Yu Maode   

  1. College of Biotechnology, Southwest University Chongqing 400715
  • Received:2014-06-27 Revised:2014-10-12 Online:2015-04-25 Published:2015-05-20

摘要:

【目的】 桑树乳胶蛋白基因在抗虫防御过程中起着重要的作用。从桑树基因组数据库中鉴定桑树 MLX56 基因家族,并进行基因进化、基因结构及基因的表达分析,为桑树 MLX56 基因的功能研究及利用奠定基础。【方法】 利用桑树基因组数据库,采用生物信息学方法,分析桑树 MLX56 基因家族结构及进化关系,并对 MLX56 基因家族成员进行鉴定; 利用MEGA4.1软件进行系统进化树分析; 通过半定量RT-PCR技术研究 MLX56 基因在桑树不同种及不同组织之间的表达水平,构建原核表达载体pET-28a-MLX56-6,并将其转入大肠杆菌BL21(DE3)中,IPTG诱导融合蛋白表达,分别收集不同诱导时间段的菌液,SDS-PAGE检测融合蛋白的表达情况。将高效表达时段的菌液进行超声破碎,SDS-PAGE检测目的蛋白的可溶性; 利用Western Blot印迹验证目的蛋白的表达。并研究该基因对大肠杆菌生长速率的影响。【结果】 利用桑树基因组数据库,鉴定6个 MLX56 家族基因,该家族基因含有2个几丁质结合域,且都含有信号肽,属于分泌蛋白。此外还克隆到1个新的 MLX56 基因,命名为 MLX56-7 (GenBank登录号为KJ496133)。系统进化树显示桑树 MLX56 基因与西洋接骨木类橡胶蛋白同源性最高,同源性达66%,与茶树几丁质酶、葡萄几丁质酶同源性较低,同源性分别为49%和48%。半定量RT-PCR分析表明, MLX56-1,MLX56-2,MLX56-4,MLX56-5,MLX56-6和MLX56-7 在桑树不同种中均有表达,组织表达特异性分析表明 MLX56-2,MLX56-4,MLX56-5,MLX56-6和MLX56-7 基因在广东桑'桂优62号'各个组织中都有表达, MLX56-1基因仅在叶柄及茎中表达,MLX56-3 基因在桑树不同种及广东桑'桂优62号'各个组织中都没有检测到表达。原核表达结果表明,MLX56-6蛋白在终浓度为 0.5 mmol ·L-1 的 IPTG 诱导下成功表达; 融合蛋白的可溶性检测表明该蛋白主要以包涵体的形式存在; Western Blot印迹检测也证实大肠杆菌BL21(DE3)中表达了1个分子量为56 kDa的蛋白。但在诱导表达过程中,大肠杆菌BL21(DE3)生长速率受到 MLX56-6 基因的抑制。【结论】 MLX56 基因家族在桑树进化过程中发生了基因的重复,且在结构上出现了分化。根据 MLX56 基因家族的蛋白及结构特征,该基因家族可能属于具有凝集素活性的几丁质酶。 MLX56 基因在桑树不同种及不同组织中的表达存在差异,暗示这些基因在桑树不同种及不同组织中的功能存在一定差异。

关键词: 桑树, MLX56 基因家族, 系统进化, 基因表达

Abstract:

【Objective】 Mulberry(Morus) latex gene plays an important role in determining anti-insect and defense. Identification the MLX56 gene family from the mulberry genome database, and analysis of phylogeny, gene structure and gene expression in mulberry will be helpful to study the functions of plant latex genes.【Method】Based on mulberry genome database, bioinformatics approach was used to analyze the structure and evolution of mulberry MLX 56 gene family, a phylogenetic tree was created using the MEGA4.1 program. The expression of MLX 56 gene in different mulberry species and different tissues were analyzed using semi-quantitative TR-PCR. A recombinant plasmid pET-28a-MLX56-6 was constructed and transferred into Escherichia coli BL21 (DE3). IPTG was used to induce MLX56-6 protein expression. Samples were collected from bacterial suspension at different times of induction and SDS-PAGE was used to analyze the protein expressed by E. coli BL21 (DE3). Ultrasonic wave was used to break the efficiently expressed bacterial suspension, and SDS-PAGE was used to detect the solubility of MLX56-6 protein. Western Blot confirmed the successful expression of MLX 56-6 in E. coli BL21 (DE3), and the effect of the MLX 56 gene on the growth rate of E. coli was also studied.【Result】A total of 6 MLX 56 genes were identified from mulberry genome database, the mulberry MLX 56 contains two chitin-binding domains, and they all have signal peptide, belongs to secretory protein. A new MLX 56 gene MLX 56-7 (GenBank number:KJ496133) was cloned. Phylogenetic analysis revealed that the highest homdogy (66%) was between mulberry MLX 56 gene and Sambucus nigra hevein-like protein, and a lower value (49%, 48%) between mulberry MLX 56 and Camellia sinensis chitinase and Vitis vinifera chitinase. The semi-quantitative RT-PCR showed that MLX56-1, MLX56-2, MLX56-4, MLX56-5, MLX56-6 and MLX56-7 were expressed in all mulberry species. Tissue specific expression analysis revealed that MLX56-2, MLX56-4, MLX56-5, MLX56-6 and MLX56-7 were expressed in all tissues of M. atropurpurea'Guiyou62', MLX56-1 was expressed only in petioles and stems. MLX56-3 gene was not detected in mulberry species and tissues in M. atropurpurea 'Guiyou62'. Prokaryotic expression results showed that the fusion protein was successfully expressed with 0.5 mmol ·L-1 IPTG induction. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western Blot confirmed that the molecular weight of the recombinant MLX56-6 was about 56 kDa. But the E.coli growth rate was inhibited by MLX56-6 gene.【Conclusion】 In the process of mulberry evolution, gene duplication happened in MLX56 gene family, and differentiation occurred in the structure of the gene family. Accordiong to the protein and structure characteristics of MLX56 gene family, the gene family may belong to a chitinase with lectin activity. The diversity among different species and tissues in MLX56 gene expression reveals that the functions of these genes were different among different mulberry species and among different tissues of the same species.

Key words: Morus, MLX56 gene family, phylogeny analysis, gene expression

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