土壤宏基因组文库PKS基因的筛选及其代谢物杀线虫活性测定
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摘要
土壤是微生物最主要的栖息地,据估计土壤中有4-5×1030个微生物细胞。然而,由于99%的微生物不能培养,所以难以得到非培养微生物的全部信息。宏基因组方法克服传统微生物培养的局限性,能够充分挖掘和利用土壤非培养微生物基因资源。本研究采用宏基因组方法,从西藏高寒草甸土壤、华北温室黄瓜地土壤微生物中筛选具有杀线虫活性的基因。
1.土壤微生物总DNA的提取方法比较
由于土壤含有丰富的有机质和腐植酸,微生物DNA提取和纯化极其困难,采用直接法和间接法提取土壤微生物总DNA,并比较不同土样的的产率以及纯度的,经电泳纯化法后更适合于构建大插入片段文库。另外,发现土壤pH、有效P、有效K及有机质会影响DNA的产率。
2.西藏高寒草甸土壤、华北温室黄瓜地土壤微生物PKS KS和NRPS A基因的筛选和功能分析
采用直接法提取的2种土壤微生物总DNA,简并引物进行PCR扩增,对土壤微生物的PKS和NRPS基因家族多样性进行研究,结果表明：从2种土壤微生物总DNA中共计得到37个PKS-KS基因(草甸土样28个、温室土样9个),40个NRPS-A基因(草甸土样24、温室土样16个)。经过BLASTX比对发现这些基因与已知的PKS(相似性57-82%)和NRPS(相似性47-67%)基因相似性较低,表明它们为新基因类型。
3.西藏高寒草甸土壤、华北温室黄瓜地土壤微生物宏基因组Fosmid文库构建
采用间接法提取和纯化2种土壤微生物总DNA,构建西藏米拉山高原草甸土壤、华北温室黄瓜地土壤微生物宏基因组Fosmid文库。西藏土壤Fosmid文库包含30 624个克隆,华北温室土壤Fosmid文库包含31 008个克隆,克隆的平均插入片段为30 kb；文库克隆稳定,无插入片段丢失或重排。通过末端测序分析发现华北温室土壤Fosmid文库中无同源序列占92.75%,而西藏土壤Fosmid文库中无同源序列占94.3%。这表明2个文库包含大量未知微生物物种,从而为挖掘新的功能基因研究奠定基础。
4.含PKS基因克隆筛选及其杀线虫活性测定
设计PKS基因KS域的简并引物,从2个土壤宏基因组文库克隆中进行PCR扩增序列筛选,共计得到7个含PKS基因的克隆。从华北温室文库获得的4个含PKS基因克隆,其PKS基因片段的长度为693 bp、699 bp、699 bp、698 bp。从西藏文库得到3个含PKS基因的克隆,其中K99携带有2个KS基因。
对筛选的PKS基因克隆经LB液体培养基培养4 d,以培养菌液浸泡线虫法测定对南方根结线虫J2、松材线虫J2的致死活性,结果表明克隆K99的杀线活性最强,其培养菌液处理12 h对两种靶标线虫的致死率为100%。温室盆栽实验表明,克隆K99对南方根结线虫的防效为89%。
5.克隆K99杀线虫活性物质的研究
通过正丁醇萃取克隆K99培养菌液和甲醇萃取菌体来检测克隆K99的活性物质,结果表明,培养菌液和菌体粗提物对松材线虫的12 h致死率分别为100%和96%。粗提物经121℃处理20min后活性不变,表明活性物质的热稳定性良好。用薄层层析追踪法表明：极性中等的C1、C2、C3条带均有杀线虫活性,而极性小的C5和极性大的C4无活性。这表明克隆K99的杀线虫活性物质可能为一种极性中等、热稳定性好的聚酮类物质。
6.一株含PKS基因放线菌杀线虫活性鉴定
根结线虫拮抗放线菌F001的发酵液对松材线虫也有致死活性,采用PCR的方法验证其是否PKS KS域基因和NRPS A域基因,并优化其发酵条件。结果表明,F001基因组中含有PKS基因和NR S基因,经Blastx比对,与已知的放线菌的相关基因相似性较高；得到最佳产杀线活性物质的培养条件是培养温度28℃、摇床转速180 r／min、装液量200mL(250 mL三角瓶)、初始pH7.0培养9 d。
总之,从2个宏基因组文库中获得多个具有杀线虫活性的克隆,尤其是克隆K99具有较高的活性,其代谢产物为极性中等,且热稳定性良好的聚酮类物质。这值得进一步研究开发。
Screening of PKS gene from Soil Metagenomics Library and Identification of the Active Substance against Nematodes
The soil is the most main habitats for microorganisms with estimated numbers of 4-5×1030 in per gram soil. However, it is difficult to obtain the whole information of unculturable microorganisms, due to over 99% of microorganisms could not be cultured by the traditional culturable methods. Metagenome break through the limitation of culturabe method, and bring a new technology for utilization for resources of non-culturable microorginasm in soil. In this papper, genes with nematocidal activity were screened from soil metagnomic library derived from Tibet and greenhouse in Huabei.
1. Comparison of methods for DNA extraction from soil
Because of amount of organic matter and humic acid in soil, it is extremely difficult to extract and purify microbial DNA from soil. Comparison of various methods for extraction and purification of DNA showed that DNA of direct extraction were short fragments and large amounts, contained high impurities and fitted for analysis of microbial diversity. However, DNA of indirect extraction that combined with differential centrifugation and Nycodenz density gradient centrifugation were long fragments, high purity and fitted for construction of large insert fragment library after purified with gel electrophoresis. In addition, pH, available P, available K and organic matter in soil had influences to DNA quantities.
2. Screening and functional analysis of PKS and NRPS from Tibet alpine meadow soil and Huabei
The diversity of family of PKS gene and NRPS gene were revealed by PCR method with degenerate primer from DNA diectly extacted from two soil samples. The result showed that 37 PKS-KS genes (28 from Tibet,9 from north China) and 40 NRPS-A genes (24 from Tibet,16 from north China) were obtained and these genes have lower similarity with knowed genes by BLASTX (similarity to PKS from 57% to 82%, similarity to NRPS from 47% to 67%), which indicated that they were probably new genes.
3. Construction of metagenomic fosmid library from Tibet and Huabei
Two metagenomic fosmid libraries were constructed using microbial DNA indirect extracted from 2 soil samples. Fosmid library from Tibet contained 30 624 clones and Fosmid library the soil example from Huabei contained 31 008 clones. The average length of insert fragments were 30 kb, and the stability of that was good without losing or resetting. The results of end sequencing of clones indicated that the percent of no significant homo logy sequences in library of the soil example from Huabei were 92.75%, yet that in library of Tibet were 94.3%. This showed that the two libraries contained many unknowed microbial speics and would lay the foundation for exploiting novel functional gene from soil.
4. Screening for clones contained PKS gene and evaluation of its nematictdal activity
A total of 7 clones contained PKS gene were obtained from two metagenomic libraries by PCR sequence selection according to degenerate primer designed from KS domain of PKS gene (4 from Huabei library,3 from Tibet library), of which K99 clone contained 2 domains. The nematocidal activity for root knot nematode and pine wood namtode was evaluated by steeping method with cultural liquid of clones for 4 days. The result showed that the nematocidal activity of clone K99 was high than others, and the rate of knockdown to two speices nematode treated with cultured liquid of clone K99 for 12 hours was 100%. The result of pot culture experiment in greenhouse also revealed that control efficiency of clone K99 against root knot nematode was 89%.
5. Identification of nematocidal active substance produced by clone K99
The lethallty rate of pine wood nematode treated with crude extact from culture liquid by normal butanol and bacterial cell of clone K99 by methanol for 12 hours were 100% and 96%, respectively. The activity of crude extract treated with hot at 121℃for 20 minuties was unchanging, which implied that the active substance was thermal stability. The result of tracking method by thin-layer chromatography showed that the channels with medium polar including channels 1, channels 2 and channels 3 possessed nematocidal activity, but the channel 5 with small polar and channel 4 with more polar did not kill nematode. From the results above, it was draw some conclusions that the active substance produced by clone K99 probably was polyketides with medium polar and thermal stability.
6. Evaluation of nematictdal activity of one strain actinomyces contained PKS gene
PKS gene and NRPS gene were amplified from one strain actinomyces F001 with nematocidal activity and the ferment condition for F001 was optimized. The result indicated that PKS gene and NRPS gene from F001 had high similarity to know gene from others actinomyces. The optimal ferment condition for F001 was culture under 28℃for 9 days, pH of medium was 7.0, rotational speed was 180 r/min and the liquid volume was 200 mL in 250 mL.
In a conclusion, some clones with nematocidal activity were obtained from two metagenomic fosmid libraries, especially clone K99 with high nematocidal activity in vivo and in pot culture experiment in greenhouse. The active substance produced by clone K99 probably was polyketides with medium polar and thermal stability. It was worth to further study.
1.土壤微生物总DNA的提取方法比较
由于土壤含有丰富的有机质和腐植酸,微生物DNA提取和纯化极其困难,采用直接法和间接法提取土壤微生物总DNA,并比较不同土样的的产率以及纯度的,经电泳纯化法后更适合于构建大插入片段文库。另外,发现土壤pH、有效P、有效K及有机质会影响DNA的产率。
2.西藏高寒草甸土壤、华北温室黄瓜地土壤微生物PKS KS和NRPS A基因的筛选和功能分析
采用直接法提取的2种土壤微生物总DNA,简并引物进行PCR扩增,对土壤微生物的PKS和NRPS基因家族多样性进行研究,结果表明：从2种土壤微生物总DNA中共计得到37个PKS-KS基因(草甸土样28个、温室土样9个),40个NRPS-A基因(草甸土样24、温室土样16个)。经过BLASTX比对发现这些基因与已知的PKS(相似性57-82%)和NRPS(相似性47-67%)基因相似性较低,表明它们为新基因类型。
3.西藏高寒草甸土壤、华北温室黄瓜地土壤微生物宏基因组Fosmid文库构建
采用间接法提取和纯化2种土壤微生物总DNA,构建西藏米拉山高原草甸土壤、华北温室黄瓜地土壤微生物宏基因组Fosmid文库。西藏土壤Fosmid文库包含30 624个克隆,华北温室土壤Fosmid文库包含31 008个克隆,克隆的平均插入片段为30 kb；文库克隆稳定,无插入片段丢失或重排。通过末端测序分析发现华北温室土壤Fosmid文库中无同源序列占92.75%,而西藏土壤Fosmid文库中无同源序列占94.3%。这表明2个文库包含大量未知微生物物种,从而为挖掘新的功能基因研究奠定基础。
4.含PKS基因克隆筛选及其杀线虫活性测定
设计PKS基因KS域的简并引物,从2个土壤宏基因组文库克隆中进行PCR扩增序列筛选,共计得到7个含PKS基因的克隆。从华北温室文库获得的4个含PKS基因克隆,其PKS基因片段的长度为693 bp、699 bp、699 bp、698 bp。从西藏文库得到3个含PKS基因的克隆,其中K99携带有2个KS基因。
对筛选的PKS基因克隆经LB液体培养基培养4 d,以培养菌液浸泡线虫法测定对南方根结线虫J2、松材线虫J2的致死活性,结果表明克隆K99的杀线活性最强,其培养菌液处理12 h对两种靶标线虫的致死率为100%。温室盆栽实验表明,克隆K99对南方根结线虫的防效为89%。
5.克隆K99杀线虫活性物质的研究
通过正丁醇萃取克隆K99培养菌液和甲醇萃取菌体来检测克隆K99的活性物质,结果表明,培养菌液和菌体粗提物对松材线虫的12 h致死率分别为100%和96%。粗提物经121℃处理20min后活性不变,表明活性物质的热稳定性良好。用薄层层析追踪法表明：极性中等的C1、C2、C3条带均有杀线虫活性,而极性小的C5和极性大的C4无活性。这表明克隆K99的杀线虫活性物质可能为一种极性中等、热稳定性好的聚酮类物质。
6.一株含PKS基因放线菌杀线虫活性鉴定
根结线虫拮抗放线菌F001的发酵液对松材线虫也有致死活性,采用PCR的方法验证其是否PKS KS域基因和NRPS A域基因,并优化其发酵条件。结果表明,F001基因组中含有PKS基因和NR S基因,经Blastx比对,与已知的放线菌的相关基因相似性较高；得到最佳产杀线活性物质的培养条件是培养温度28℃、摇床转速180 r／min、装液量200mL(250 mL三角瓶)、初始pH7.0培养9 d。
总之,从2个宏基因组文库中获得多个具有杀线虫活性的克隆,尤其是克隆K99具有较高的活性,其代谢产物为极性中等,且热稳定性良好的聚酮类物质。这值得进一步研究开发。
Screening of PKS gene from Soil Metagenomics Library and Identification of the Active Substance against Nematodes
The soil is the most main habitats for microorganisms with estimated numbers of 4-5×1030 in per gram soil. However, it is difficult to obtain the whole information of unculturable microorganisms, due to over 99% of microorganisms could not be cultured by the traditional culturable methods. Metagenome break through the limitation of culturabe method, and bring a new technology for utilization for resources of non-culturable microorginasm in soil. In this papper, genes with nematocidal activity were screened from soil metagnomic library derived from Tibet and greenhouse in Huabei.
1. Comparison of methods for DNA extraction from soil
Because of amount of organic matter and humic acid in soil, it is extremely difficult to extract and purify microbial DNA from soil. Comparison of various methods for extraction and purification of DNA showed that DNA of direct extraction were short fragments and large amounts, contained high impurities and fitted for analysis of microbial diversity. However, DNA of indirect extraction that combined with differential centrifugation and Nycodenz density gradient centrifugation were long fragments, high purity and fitted for construction of large insert fragment library after purified with gel electrophoresis. In addition, pH, available P, available K and organic matter in soil had influences to DNA quantities.
2. Screening and functional analysis of PKS and NRPS from Tibet alpine meadow soil and Huabei
The diversity of family of PKS gene and NRPS gene were revealed by PCR method with degenerate primer from DNA diectly extacted from two soil samples. The result showed that 37 PKS-KS genes (28 from Tibet,9 from north China) and 40 NRPS-A genes (24 from Tibet,16 from north China) were obtained and these genes have lower similarity with knowed genes by BLASTX (similarity to PKS from 57% to 82%, similarity to NRPS from 47% to 67%), which indicated that they were probably new genes.
3. Construction of metagenomic fosmid library from Tibet and Huabei
Two metagenomic fosmid libraries were constructed using microbial DNA indirect extracted from 2 soil samples. Fosmid library from Tibet contained 30 624 clones and Fosmid library the soil example from Huabei contained 31 008 clones. The average length of insert fragments were 30 kb, and the stability of that was good without losing or resetting. The results of end sequencing of clones indicated that the percent of no significant homo logy sequences in library of the soil example from Huabei were 92.75%, yet that in library of Tibet were 94.3%. This showed that the two libraries contained many unknowed microbial speics and would lay the foundation for exploiting novel functional gene from soil.
4. Screening for clones contained PKS gene and evaluation of its nematictdal activity
A total of 7 clones contained PKS gene were obtained from two metagenomic libraries by PCR sequence selection according to degenerate primer designed from KS domain of PKS gene (4 from Huabei library,3 from Tibet library), of which K99 clone contained 2 domains. The nematocidal activity for root knot nematode and pine wood namtode was evaluated by steeping method with cultural liquid of clones for 4 days. The result showed that the nematocidal activity of clone K99 was high than others, and the rate of knockdown to two speices nematode treated with cultured liquid of clone K99 for 12 hours was 100%. The result of pot culture experiment in greenhouse also revealed that control efficiency of clone K99 against root knot nematode was 89%.
5. Identification of nematocidal active substance produced by clone K99
The lethallty rate of pine wood nematode treated with crude extact from culture liquid by normal butanol and bacterial cell of clone K99 by methanol for 12 hours were 100% and 96%, respectively. The activity of crude extract treated with hot at 121℃for 20 minuties was unchanging, which implied that the active substance was thermal stability. The result of tracking method by thin-layer chromatography showed that the channels with medium polar including channels 1, channels 2 and channels 3 possessed nematocidal activity, but the channel 5 with small polar and channel 4 with more polar did not kill nematode. From the results above, it was draw some conclusions that the active substance produced by clone K99 probably was polyketides with medium polar and thermal stability.
6. Evaluation of nematictdal activity of one strain actinomyces contained PKS gene
PKS gene and NRPS gene were amplified from one strain actinomyces F001 with nematocidal activity and the ferment condition for F001 was optimized. The result indicated that PKS gene and NRPS gene from F001 had high similarity to know gene from others actinomyces. The optimal ferment condition for F001 was culture under 28℃for 9 days, pH of medium was 7.0, rotational speed was 180 r/min and the liquid volume was 200 mL in 250 mL.
In a conclusion, some clones with nematocidal activity were obtained from two metagenomic fosmid libraries, especially clone K99 with high nematocidal activity in vivo and in pot culture experiment in greenhouse. The active substance produced by clone K99 probably was polyketides with medium polar and thermal stability. It was worth to further study.
引文
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