内源性抗菌肽基因的过量表达对家蚕抗病力影响的研究
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摘要
昆虫抗菌肽是昆虫免疫诱导产生的一系列小分子多肽,对昆虫的抗病能力起着重要的作用。昆虫抗菌肽具有杀菌能力强、抗菌谱广等特点,而引起人们的普遍关注。家蚕作为支撑蚕丝产业的重要经济昆虫,因此提高其抗病性的研究对提高蚕丝生产有着重要的意义。家蚕是鳞翅目昆虫中的模式生物,国内外研究者已经对家蚕的抗菌肽做了大量的研究工作,发现家蚕中存Cecropin、Moricin、Attacin、Defencin、Gloverin、Lebocin这六大类抗菌肽基因,这些基因对家蚕的抗病能力应该发挥着重要的作用。近年来,随着基因工程技术的发展,通过转基因手段培育抗病性强的新品种成为新的育种研究方向,人们把昆虫抗菌肽基因导入动植物染色体,已经获得了抗病性强的转基因动植物,为选育高抗性的动植物品种作出了重要的探索。
本研究基于本实验室构建的家蚕转基因技术平台,选择对革兰氏阳性菌与革兰氏阴性菌有抗菌活性的家蚕Moricin2基因(NCBI LOCUS:AB006915.1)和对革兰氏阴性菌有抗菌活性的家蚕Lebocin3基因(NCBI LOCUS:AB003035),构建转基因表达载体并导入家蚕基因组中,探索转内源性抗菌肽基因后对家蚕抗菌能力和生长发育的影响。本研究获得的如下结果:
1.抗菌肽转基因表达载体的构建
将主要在家蚕脂肪体表达的SDH启动子与Morcin2基因构建成融合转基因表达载体pBac[3×P3-EGFP+SDH-Mor];将Lebocin3基因分别与SDH启动子或在家蚕全身广谱表达的IE1启动子构建成两个融合转基因表达载体,即pBac[3×P3-DsRed+IE1-Leb]和pBac[3×P3-DsRed+SDH-Leb]。
2.转基因蚕的制作与分子检测
将构建好的pBac[3×P3-EGFP+SDH-Mor]、pBac[3×P3-DsRed+IE1-Leb]和pBac[3×P3-DsRed+SDH-Leb]3个转基因表达载体通过蚕卵显微注射后,在G1代进行荧光筛选,分别得到40、30、36头荧光蚕。
对每个阳性转基因家蚕品系随机抽取一个个体进行Southern blot检测,分析结果表明,目的基因表达盒都已经整合到家蚕基因组上,即成功构建了SDH-Mor、IE1-Leb和SDH-Leb转基因系。
3.转基因家蚕中Moricin2和Lebocin3抗菌肽基因的表达分析
抽提3个转基因系和对照的脂肪体总RNA,合成cDNA后进行半定量RT-PCR检测,结果表明:(1) SDH-Mor和对照脂肪体中Moricin2基因mRNA的相对表达量分别为0.72±0.018和0.43±0.0093,Moricin2基因mRNA相对表达量提高差异极显著;(2) IE1-Leb、SDH-Leb和对照脂肪体中Lebocin3基因mRNA的相对表达量分别是0.73±0.015、0.71±0.017和0.42±0.0091,两个转基因系中Lebocin3基因mRNA的相对表达量比对照显著提高,但是两个转基因系间差异不显著。
4.转基因蚕的抗菌能力分析和生长发育情况观察
对SDH-Mor转基因系和对照2龄起蚕添食苏云金芽孢杆菌,试验结果表明,当苏云金芽孢杆菌浓度为1.25x10~8个/mL时,SDH-Mor转基因系比对照对苏云金芽孢杆菌的抗性提高了约24.4%。
对IE1-Leb转基因系和SDH-Leb转基因系与对照2龄起蚕添食E.coli,实验结果显示,当E.col浓度为1.4×10~9个/mL时,IE1-Leb转基因系和SDH-Leb转基因系对E.coli的抗性比对照分别提高了约20.57%和13.9%。
对转基因家蚕的生长发育情况进行观察,没有发现明显的异常现象。
The insect antibacterial peptide is small-molecule polypeptides which plays a very important role in the process of insect resistance.Because of its strong antibactericidal capacity and wide antibacterial spectrum,it attracts attention commonly.Domesticated silkworms,Bomyx mori,is important economic insect which surports the silk industry. It is meaningful for enhancing the resistance to increase production of silk.At the same time,Domesticated silkworms,Bomyx mori,is the typical model organisms in Lepidoptera,many scholars have been done lots of researches and founded 6 types antibacterial peptide of silkworm.In recent years,with the development of the transgenic technology,the experiment of increasing the resistance by importing the gene of antibacterial peptide into the genome of animals and plants has been done successfully.
We choosed silkworm Moricin 2 and Lebocin 3,and constructed the transgenic expression vector with different promoters.The early embryos were microinjected with the vectors,and detection transgenic silkworm by Florescencing and Southern blot,and analysis the relative expression mRNA of Moricin 2 and Lebocin 3,and then exploreing the influence of transfered endogenous antibaicterial peptide gene on the resistance and growth of silkworm.The main research results are as follows:
1.The Construction of Transgenic Expression Vector
We constructed transgenic expression vector pBac[3×P3-EGFP+SDH-Mor]in which Morcin2 gene was controled by the SDH promoter which is mainly expressed in the silkworm fat body; Lebocin 3 transgenic expression vectors pBac[3×P3-DsRed+IE1-Leb]and pBac[3×P3-DsRed+ SDH-Leb]constructed with different promoter.
2.Generation and detection transgenic silkworm
Silkworm eggs were microinjected with the pBac[3×P3-EGFP+SDH-Mor]、pBac[3×P3-DsRed+IE1-Leb]and pBac[3×P3-DsRed+SDH-Leb]expression vectors respepctively.40,30 and 36 transgenic positive individuals were obtained in G1 generation respectively.Southern blot assay showed that all of expression boxes are inserted into chromosome of silkworm.
3.Expression analysis of Moricin 2 and Lebocin 3 in the transgenic silkworm
The semi-quantitative RT-PCR showed,(1) mRNA of Moricin2 gene relative expression value of SDH-Mor transgenic line and the control was 0.72±0.018 and 0.43±0.0093 respectively,the difference is significant;(2) mRNA of Lebocin3 gene relative expression value of IE1-Leb and SDH-Leb transgenic line and the control was 0.73±0.015,0.71±0.017 and 0.42±0.0091 respectively,and the difference of transgenic lines and control is significant,but that of between IE1-Leb and SDH-Leb transgenic line is not.
4.Resistance analysis of transgenic silkworm
2 instar larva of SDH-Mor transgenic silkworm and control reared with diet which treated with the Bacillus thuringiensis.The results showed that when the concentration of Bacillus thuringiensi is up to 1.25×10~8 per mL,the resistance of SDH-Mor transgenic strains is increase about 24.4%than that of control.
2 instar larva of IE1-Leb、SDH-Leb transgenic silkworm and control reared with diet which added E.coli,the results indicated that when the concentration of E.coli is up to 1.4×10~9 per mL,the resistance of IE1-Leb and SDH-Leb transgenic strains is raise about 20.57%and 13.9%respectively.
We did not observe any abnormal about the growth of transgenic silkworm.
本研究基于本实验室构建的家蚕转基因技术平台,选择对革兰氏阳性菌与革兰氏阴性菌有抗菌活性的家蚕Moricin2基因(NCBI LOCUS:AB006915.1)和对革兰氏阴性菌有抗菌活性的家蚕Lebocin3基因(NCBI LOCUS:AB003035),构建转基因表达载体并导入家蚕基因组中,探索转内源性抗菌肽基因后对家蚕抗菌能力和生长发育的影响。本研究获得的如下结果:
1.抗菌肽转基因表达载体的构建
将主要在家蚕脂肪体表达的SDH启动子与Morcin2基因构建成融合转基因表达载体pBac[3×P3-EGFP+SDH-Mor];将Lebocin3基因分别与SDH启动子或在家蚕全身广谱表达的IE1启动子构建成两个融合转基因表达载体,即pBac[3×P3-DsRed+IE1-Leb]和pBac[3×P3-DsRed+SDH-Leb]。
2.转基因蚕的制作与分子检测
将构建好的pBac[3×P3-EGFP+SDH-Mor]、pBac[3×P3-DsRed+IE1-Leb]和pBac[3×P3-DsRed+SDH-Leb]3个转基因表达载体通过蚕卵显微注射后,在G1代进行荧光筛选,分别得到40、30、36头荧光蚕。
对每个阳性转基因家蚕品系随机抽取一个个体进行Southern blot检测,分析结果表明,目的基因表达盒都已经整合到家蚕基因组上,即成功构建了SDH-Mor、IE1-Leb和SDH-Leb转基因系。
3.转基因家蚕中Moricin2和Lebocin3抗菌肽基因的表达分析
抽提3个转基因系和对照的脂肪体总RNA,合成cDNA后进行半定量RT-PCR检测,结果表明:(1) SDH-Mor和对照脂肪体中Moricin2基因mRNA的相对表达量分别为0.72±0.018和0.43±0.0093,Moricin2基因mRNA相对表达量提高差异极显著;(2) IE1-Leb、SDH-Leb和对照脂肪体中Lebocin3基因mRNA的相对表达量分别是0.73±0.015、0.71±0.017和0.42±0.0091,两个转基因系中Lebocin3基因mRNA的相对表达量比对照显著提高,但是两个转基因系间差异不显著。
4.转基因蚕的抗菌能力分析和生长发育情况观察
对SDH-Mor转基因系和对照2龄起蚕添食苏云金芽孢杆菌,试验结果表明,当苏云金芽孢杆菌浓度为1.25x10~8个/mL时,SDH-Mor转基因系比对照对苏云金芽孢杆菌的抗性提高了约24.4%。
对IE1-Leb转基因系和SDH-Leb转基因系与对照2龄起蚕添食E.coli,实验结果显示,当E.col浓度为1.4×10~9个/mL时,IE1-Leb转基因系和SDH-Leb转基因系对E.coli的抗性比对照分别提高了约20.57%和13.9%。
对转基因家蚕的生长发育情况进行观察,没有发现明显的异常现象。
The insect antibacterial peptide is small-molecule polypeptides which plays a very important role in the process of insect resistance.Because of its strong antibactericidal capacity and wide antibacterial spectrum,it attracts attention commonly.Domesticated silkworms,Bomyx mori,is important economic insect which surports the silk industry. It is meaningful for enhancing the resistance to increase production of silk.At the same time,Domesticated silkworms,Bomyx mori,is the typical model organisms in Lepidoptera,many scholars have been done lots of researches and founded 6 types antibacterial peptide of silkworm.In recent years,with the development of the transgenic technology,the experiment of increasing the resistance by importing the gene of antibacterial peptide into the genome of animals and plants has been done successfully.
We choosed silkworm Moricin 2 and Lebocin 3,and constructed the transgenic expression vector with different promoters.The early embryos were microinjected with the vectors,and detection transgenic silkworm by Florescencing and Southern blot,and analysis the relative expression mRNA of Moricin 2 and Lebocin 3,and then exploreing the influence of transfered endogenous antibaicterial peptide gene on the resistance and growth of silkworm.The main research results are as follows:
1.The Construction of Transgenic Expression Vector
We constructed transgenic expression vector pBac[3×P3-EGFP+SDH-Mor]in which Morcin2 gene was controled by the SDH promoter which is mainly expressed in the silkworm fat body; Lebocin 3 transgenic expression vectors pBac[3×P3-DsRed+IE1-Leb]and pBac[3×P3-DsRed+ SDH-Leb]constructed with different promoter.
2.Generation and detection transgenic silkworm
Silkworm eggs were microinjected with the pBac[3×P3-EGFP+SDH-Mor]、pBac[3×P3-DsRed+IE1-Leb]and pBac[3×P3-DsRed+SDH-Leb]expression vectors respepctively.40,30 and 36 transgenic positive individuals were obtained in G1 generation respectively.Southern blot assay showed that all of expression boxes are inserted into chromosome of silkworm.
3.Expression analysis of Moricin 2 and Lebocin 3 in the transgenic silkworm
The semi-quantitative RT-PCR showed,(1) mRNA of Moricin2 gene relative expression value of SDH-Mor transgenic line and the control was 0.72±0.018 and 0.43±0.0093 respectively,the difference is significant;(2) mRNA of Lebocin3 gene relative expression value of IE1-Leb and SDH-Leb transgenic line and the control was 0.73±0.015,0.71±0.017 and 0.42±0.0091 respectively,and the difference of transgenic lines and control is significant,but that of between IE1-Leb and SDH-Leb transgenic line is not.
4.Resistance analysis of transgenic silkworm
2 instar larva of SDH-Mor transgenic silkworm and control reared with diet which treated with the Bacillus thuringiensis.The results showed that when the concentration of Bacillus thuringiensi is up to 1.25×10~8 per mL,the resistance of SDH-Mor transgenic strains is increase about 24.4%than that of control.
2 instar larva of IE1-Leb、SDH-Leb transgenic silkworm and control reared with diet which added E.coli,the results indicated that when the concentration of E.coli is up to 1.4×10~9 per mL,the resistance of IE1-Leb and SDH-Leb transgenic strains is raise about 20.57%and 13.9%respectively.
We did not observe any abnormal about the growth of transgenic silkworm.
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60.马志明,石晓燕,曹广力,薛仁宇,何毅,贡成良.家蚕核型多角体病毒IE—1基因启动子的克隆和序列分析.江苏蚕业.2001,23(2):1-6
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66.郭丽,王天云,王俐,咎玉玺,杨保胜.半定量反转录-聚合酶链反应分析大鼠不同组织肌肉生长抑制素基因的表达.中国组织工程研究与临床康复2008,12(11):2164-2166
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72.Hiromitsu Tanaka,Masafumi Yamamoto,Yuko Moriyama et al..A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori.Gene Structure and Expression.2005,1730(1):10-21
73.Xiaoxia Xu,Fengliang Jin,Xiaoqiang Yu et al..High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli.Ptotein Expression and Purification.2007,55(1):175-182
74.庹晓晔,柴家科.抗菌肽的基因工程研究进展生物技术通讯.2007,18(2):298-300
75.王建军,王恬.抗菌肽及其在动物生产上的应用.家畜生态学报.2008,29(3):97-101
76.祝骥,高飞,易喻.抗菌肽的研究进展.生命科学.2008,20(4):605-611
77.张祥喜,熊尚文.家蚕抗菌肽对花生青基病菌的抑制作用初报.中国油料作物学报.1998,20(2):84-86
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79.郭玉梅,戴祝英.家蚕抗菌肽的一些性质及抗肿瘤活性.南京师大学报.1995,18(1):62-68
80.刘忠渊,张富春,毛新芳.家蚕抗菌肽的特性与应用.生物技术通讯.2003,13(5):48-51
81.曾辉明,黄俊生,程立生.昆虫抗菌肽的研究进展.热带农业科学.2002,22(6):69-76
82 许兵红,陈正耀.昆虫体液免疫中的抗菌物质.昆虫知识.1999,36(5):316-320
83.Darien E.Reed,Xinhua M.Huang,James A.Wohlschlegel and et al.DEAF-1 regulates immunity gene expression in Drosophila.PNAS.2008,105(24):351-356
84.马志明,石晓燕,查新民,何毅贡,成良.转基因家蚕研究展.2001,2:6-11
85.Hara S,Yamakawa M.Cooperative antibacterial re-lationship between lebocin and cecropin D,antibacterial peptides isolated from the silkworm Bombyx mori(Lepidoptera;Bombycidae).Appl Entomol Zool.1995;30:606-618.
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