家蚕TILLING技术的优化及其在丝素基因突变体筛选中的初步应用
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摘要
随着基因组测序工作的不断推进,人类已经进入了以功能基因组研究为主题的后基因组时代,其中一个显著的特点就是,发现新基因和认识基因的生物学功能成为最为紧迫的任务。突变体是功能基因组学研究的重要材料,受到人们越来越广泛的重视。在这样的大背景下,大规模突变体库的构建就成为了功能基因组研究的一个重要内容。获得突变体的方法很多,除了收集自然突变体以外,还有采用人工诱变的方法,如物理化学诱变、基因敲除(knock-out)、基因沉默(gene silence)、插入突变(insertional mutagenesis)等,每种方法都各有其优缺点。定向诱导基因组局部突变——TILLING(Targeting Induced Local Lesions InGenomes)是近年发展起来的一种全新的反向遗传学方法。它的基本原理是:采用特异基因引物对应用化学诱变剂所产生的突变群体进行PCR放大突变信号,经变性和复性过程得到异源双链DNA分子,再用能特异切割错配位点的核酸内切酶(如CEL I)切割异源双链,并用变性电泳检测酶切产物,最后筛选出目的基因突变体。该技术具有通量高、操作简单、成本低等优点,可以有效克服其它获得突变体方法的繁琐、费时等不足,因此被广泛应用于多种生物的功能基因组学研究。我们有理由相信,TILLING技术在家蚕中也能充分体现其潜在的优势并发挥其重要的作用。
本研究在前人研究的基础上,应用化学诱变剂N-甲基N-亚硝基脲(MNU)对家蚕进行诱变处理,构建了化学诱变的家蚕突变体库;同时优化了家蚕TILLING技术的检测体系;并在此基础上,以家蚕重要的功能基因之一即丝素基因作为研究对象,初步应用TILLING技术筛选了目标区域的点突变,以期验证该技术在家蚕中应用的可行性并获得丝素基因突变体。主要结果如下:
(1)首先调查了诱变剂MNU对家蚕第五天蛹的半致死剂量(LD_(50)),结果表明,注射体积为50μL的情况下,MNU在雌雄蛹中半致死浓度分别为:雌蛹1730μg/mL、雄蛹1416.7μg/mL;即LD_(50)(雌蛹)=86.5μg/头、LD_(50)(雄蛹)=70.8μg/头。其次用MNU对家蚕进行诱变,构建了家蚕突变体库,同时调查了M_0代化蛾率和M_1代卵的孵化率,结果发现MNU诱变浓度为1000μg/mL时,M_0代化蛾率低且M_1代卵胚胎致死现象严重;MNU诱变浓度为100μg/mL时,M_0代化蛾率及M_1代卵的孵化率都明显提高,并获得了较多存活的诱变后代;另外,通过观察诱变后代的可视表型,发现在100μg/mLMNU诱导家蚕第5天蛹的M_2代中有一编号为M18-21蛾区的家蚕在幼虫发育到三眠时出现1/4致死现象,且M_3代仍呈1/4致死,推测该突变体是MNU诱变产生的隐性致死突变。
(2)应用改进后的家蚕基因组DNA提取方法,提取到815个M_1代基因组DNA样品;将这些样品排列于96孔微量板上,以单向构池法构建了2个可供TILLING检测筛选的4×DNA池,为家蚕TILLING技术的应用奠定了基础。
(3)对家蚕TILLING检测体系中PCR模板量和酶切反应体系进行了优化,结果表明:家蚕TILLING检测过程中用于PCR模板的基因组DNA最适用量为～20ng;用CEL I酶切消化异源双链核酸分子时最适孵育时间为15min,最佳酶用量为每20μL反应体系中加入0.5μL本实验室提取的CEL I酶的10倍稀释液。
(4)以丝素基因fib-l和P25为研究对象,初步应用TILLING技术对815个家蚕MNU诱变个体进行检测,其中对fib-l基因检测的碱基数量共计约为810kb,初步筛选到1个突变样C3-2和1个突变体M18-21;对P25基因检测的碱基数量共计约为1500kb,目前暂未检测到突变样。测序结果显示,在样品M18-21中,扩增子Fib-L-C的第107bp处有T和C两个信号的杂合峰,认为是碱基转换突变,即T＞T/C;由于该位点位于内含子区,不会影响编码氨基酸序列;且该突变位点与TILLING凝胶图片中酶切片段大小反映出的信息基本相符。而突变样C3-2中的具体突变个体和突变情况还有待进一步检测和验证。这些结果表明TILLING技术在家蚕中的应用是可行的。
综上所述,本研究首先建立了一个MNU诱导家蚕突变库,为TILLING技术在家蚕基因突变体筛选中提供了材料;同时,优化了家蚕TILLING技术的检测体系,为该技术进一步应用奠定了基础;另外,初步应用该技术对丝素基因突变体进行检测并测序验证,获得了1个化学诱导突变体。通过本研究,我们相信TILLING技术将为家蚕功能基因组研究提供更多有效的化学诱导突变体材料,从而推动家蚕基因功能的研究,促进蚕业科学的发展。
With the development of genomes sequencing,the focus is shiftting to discover and explore gene function in the post-genomic era.Construction of large-scale mutation library is an important part of functional genomics.Mutant populations are indispensable genetic resources for functional genomics in all organisms.There are many ways to obtain mutants,such as physical and chemical mutagenesis,gene knock-out,gene silence,insertional mutagenesis,etc,each of which has its special features.TILLING(Targeting Induced Local Lesions In Genomes)is a newly developed reverse genetic technology.Its principle is that:the experimental materials were induced by chemical mutagen and produced mutation population,then the mutation signals may be zoomed by PCR with gene special primers,after heating and annealing,heteroduplexes were recognized and cut at mismatched sites by the endonuclease(eg.CEL I),cut strands were visualized using denaturing gel electrophoresis,lastly target gene mutants can be screened.Compared with other methods this technique has many advantages such as high-throughput、easy operation、economic cost,which overcome the shortage of other mutation methods.TILLING is widely used in functional genomic researches of multiple organisms.So it is believed to have potential application in functional analysis of silkworm.
Based on the former researches,we mutagenized the silkworm pupa by chemical mutagens N-methyl-N-nitrosourea(MNU)to construct a mutation library,then we improved the detection conditions and chose fibroin genes as target for the primary application of the TILLING in silkworm.The results are as follows:
1.We investigated the lethal dosage 50%(LD_(50))of the 5th day pupa treated with MNU,the result showed that when the injection volume was 50μL,the median lethal concentration respectively was 1730μg/mL in female pupa,1416.7μg/mL in male pupa;that is LD_(50)(female)=86.5μg/pupa、LD_(50) (male)=70.8μg/pupa.We also examined the eclosion rate of M_0 generation and hatch rate of M_1 egg.When the MNU concentration was 1000μg/mL the eclosion rate was low and M_1 egg mostly dead in embryo.More live mutagenized individuals could be obtained when the concentration of MNU was 100μg/mL.The silkworm larva of M_2 generation in M18-21 exhibited a quarter-death rate in the 3rd molt,and it still remained in the M_3 generation,which suggested that a recessive lethal mutation caused by MNU might exist.
2.A high-throughput method for genomic DNA extraction was constructed.815 genomic DNA samples were obtained and arrayed in 96-well pates.Two 4×DNA pools were constructed for TILLING by one dimensional pooling method,which supplied the basis for applying TILLING in silkworm Bombyx mori.
3.Templete quantities in PCR reaction and enzyme reaction system in TILLING were optimized in this research.The current optimal reaction conditions were as follows:the optimal amount of genomic DNA for PCR reaction was～20ng,the best incubation time of heteroduplex digestion by CELI was 15min,the optimum amount of CELI was 0.51μL of diluted in ten-fold solution for every 20μL digestion reaction system.The TILLING detection system established in this study should be helpful to improve the efficiency of screening mutation of silkworm.
4.To screen the mutation of Fib-1 and P25 gene,815 MNU-mutagenized M_1 individuals were investigated by TILLING.For Fib-1,a total of 810kb were screened,and two mutations were found and named C3-2,M18-21.For P25,no mutant was found presently after screening of a total of 1500kb.The sequencing results showed that in M18-21,there are two signals in the 107th bp of amplicon Fib-1-C:T and C.The site was located in intron which could not bring change to protein coding.And also,the gel image of TILLING provided support to the mutation of this site.The mutation sample of C3-2 shoud be detected and validated farther.These had suggested that TILLING could be used in silkworm.
In conclusion,to apply TILLING to silkworm,we developed a mutagenized silkworm population by treatment with the chemical mutagen MNU.We also optimized the amount of genomic DNA template in PCR reaction and enzyme reaction system.In addition,we detected and identified Fib-1 and P25 genes mutation from mutation library by TILLING At pesent,one mutant induced by MNU was obtained.It is believed that TILLING would become a popular method applied in the functional genomic research of silkworm.
本研究在前人研究的基础上,应用化学诱变剂N-甲基N-亚硝基脲(MNU)对家蚕进行诱变处理,构建了化学诱变的家蚕突变体库;同时优化了家蚕TILLING技术的检测体系;并在此基础上,以家蚕重要的功能基因之一即丝素基因作为研究对象,初步应用TILLING技术筛选了目标区域的点突变,以期验证该技术在家蚕中应用的可行性并获得丝素基因突变体。主要结果如下:
(1)首先调查了诱变剂MNU对家蚕第五天蛹的半致死剂量(LD_(50)),结果表明,注射体积为50μL的情况下,MNU在雌雄蛹中半致死浓度分别为:雌蛹1730μg/mL、雄蛹1416.7μg/mL;即LD_(50)(雌蛹)=86.5μg/头、LD_(50)(雄蛹)=70.8μg/头。其次用MNU对家蚕进行诱变,构建了家蚕突变体库,同时调查了M_0代化蛾率和M_1代卵的孵化率,结果发现MNU诱变浓度为1000μg/mL时,M_0代化蛾率低且M_1代卵胚胎致死现象严重;MNU诱变浓度为100μg/mL时,M_0代化蛾率及M_1代卵的孵化率都明显提高,并获得了较多存活的诱变后代;另外,通过观察诱变后代的可视表型,发现在100μg/mLMNU诱导家蚕第5天蛹的M_2代中有一编号为M18-21蛾区的家蚕在幼虫发育到三眠时出现1/4致死现象,且M_3代仍呈1/4致死,推测该突变体是MNU诱变产生的隐性致死突变。
(2)应用改进后的家蚕基因组DNA提取方法,提取到815个M_1代基因组DNA样品;将这些样品排列于96孔微量板上,以单向构池法构建了2个可供TILLING检测筛选的4×DNA池,为家蚕TILLING技术的应用奠定了基础。
(3)对家蚕TILLING检测体系中PCR模板量和酶切反应体系进行了优化,结果表明:家蚕TILLING检测过程中用于PCR模板的基因组DNA最适用量为～20ng;用CEL I酶切消化异源双链核酸分子时最适孵育时间为15min,最佳酶用量为每20μL反应体系中加入0.5μL本实验室提取的CEL I酶的10倍稀释液。
(4)以丝素基因fib-l和P25为研究对象,初步应用TILLING技术对815个家蚕MNU诱变个体进行检测,其中对fib-l基因检测的碱基数量共计约为810kb,初步筛选到1个突变样C3-2和1个突变体M18-21;对P25基因检测的碱基数量共计约为1500kb,目前暂未检测到突变样。测序结果显示,在样品M18-21中,扩增子Fib-L-C的第107bp处有T和C两个信号的杂合峰,认为是碱基转换突变,即T＞T/C;由于该位点位于内含子区,不会影响编码氨基酸序列;且该突变位点与TILLING凝胶图片中酶切片段大小反映出的信息基本相符。而突变样C3-2中的具体突变个体和突变情况还有待进一步检测和验证。这些结果表明TILLING技术在家蚕中的应用是可行的。
综上所述,本研究首先建立了一个MNU诱导家蚕突变库,为TILLING技术在家蚕基因突变体筛选中提供了材料;同时,优化了家蚕TILLING技术的检测体系,为该技术进一步应用奠定了基础;另外,初步应用该技术对丝素基因突变体进行检测并测序验证,获得了1个化学诱导突变体。通过本研究,我们相信TILLING技术将为家蚕功能基因组研究提供更多有效的化学诱导突变体材料,从而推动家蚕基因功能的研究,促进蚕业科学的发展。
With the development of genomes sequencing,the focus is shiftting to discover and explore gene function in the post-genomic era.Construction of large-scale mutation library is an important part of functional genomics.Mutant populations are indispensable genetic resources for functional genomics in all organisms.There are many ways to obtain mutants,such as physical and chemical mutagenesis,gene knock-out,gene silence,insertional mutagenesis,etc,each of which has its special features.TILLING(Targeting Induced Local Lesions In Genomes)is a newly developed reverse genetic technology.Its principle is that:the experimental materials were induced by chemical mutagen and produced mutation population,then the mutation signals may be zoomed by PCR with gene special primers,after heating and annealing,heteroduplexes were recognized and cut at mismatched sites by the endonuclease(eg.CEL I),cut strands were visualized using denaturing gel electrophoresis,lastly target gene mutants can be screened.Compared with other methods this technique has many advantages such as high-throughput、easy operation、economic cost,which overcome the shortage of other mutation methods.TILLING is widely used in functional genomic researches of multiple organisms.So it is believed to have potential application in functional analysis of silkworm.
Based on the former researches,we mutagenized the silkworm pupa by chemical mutagens N-methyl-N-nitrosourea(MNU)to construct a mutation library,then we improved the detection conditions and chose fibroin genes as target for the primary application of the TILLING in silkworm.The results are as follows:
1.We investigated the lethal dosage 50%(LD_(50))of the 5th day pupa treated with MNU,the result showed that when the injection volume was 50μL,the median lethal concentration respectively was 1730μg/mL in female pupa,1416.7μg/mL in male pupa;that is LD_(50)(female)=86.5μg/pupa、LD_(50) (male)=70.8μg/pupa.We also examined the eclosion rate of M_0 generation and hatch rate of M_1 egg.When the MNU concentration was 1000μg/mL the eclosion rate was low and M_1 egg mostly dead in embryo.More live mutagenized individuals could be obtained when the concentration of MNU was 100μg/mL.The silkworm larva of M_2 generation in M18-21 exhibited a quarter-death rate in the 3rd molt,and it still remained in the M_3 generation,which suggested that a recessive lethal mutation caused by MNU might exist.
2.A high-throughput method for genomic DNA extraction was constructed.815 genomic DNA samples were obtained and arrayed in 96-well pates.Two 4×DNA pools were constructed for TILLING by one dimensional pooling method,which supplied the basis for applying TILLING in silkworm Bombyx mori.
3.Templete quantities in PCR reaction and enzyme reaction system in TILLING were optimized in this research.The current optimal reaction conditions were as follows:the optimal amount of genomic DNA for PCR reaction was～20ng,the best incubation time of heteroduplex digestion by CELI was 15min,the optimum amount of CELI was 0.51μL of diluted in ten-fold solution for every 20μL digestion reaction system.The TILLING detection system established in this study should be helpful to improve the efficiency of screening mutation of silkworm.
4.To screen the mutation of Fib-1 and P25 gene,815 MNU-mutagenized M_1 individuals were investigated by TILLING.For Fib-1,a total of 810kb were screened,and two mutations were found and named C3-2,M18-21.For P25,no mutant was found presently after screening of a total of 1500kb.The sequencing results showed that in M18-21,there are two signals in the 107th bp of amplicon Fib-1-C:T and C.The site was located in intron which could not bring change to protein coding.And also,the gel image of TILLING provided support to the mutation of this site.The mutation sample of C3-2 shoud be detected and validated farther.These had suggested that TILLING could be used in silkworm.
In conclusion,to apply TILLING to silkworm,we developed a mutagenized silkworm population by treatment with the chemical mutagen MNU.We also optimized the amount of genomic DNA template in PCR reaction and enzyme reaction system.In addition,we detected and identified Fib-1 and P25 genes mutation from mutation library by TILLING At pesent,one mutant induced by MNU was obtained.It is believed that TILLING would become a popular method applied in the functional genomic research of silkworm.
引文
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