CN108754018B - 一种刺五加目的基因ssr分子标记的筛选方法及应用 - Google Patents
一种刺五加目的基因ssr分子标记的筛选方法及应用 Download PDFInfo
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Abstract
本发明公开了一种刺五加目的基因SSR分子标记的筛选方法及应用，筛选方法具体步骤为：1)分别提取刺五加各组织的总RNA；2)总RNA反转录后进行RNA‑seq测序，Trinity软件进行unigene组装；3)筛选出与刺五加皂苷、SOD等合成积累相关的unigene，MicroSatellite软件筛选这些unigene上的SSR位点；4)Primer3.0软件设计引物，并合成；5)基因组DNA为模板进行PCR扩增；6)PCR产物进行电泳检测，开发目的基因SSR引物。本发明高效、更有针对性的开发与目的性状相关的候选SSR分子标记，为优良刺五加种质资源的分子鉴定提供有效标记。对优良刺五加种质资源早期鉴定和加快育种进程具有重要意义。
Description
技术领域
本发明属于刺五加分子标记开发、分子生物技术领域，本发明涉及一种刺五加目的基因SSR分子标记的筛选方法及应用。
背景技术
刺五加为五加科五加属落叶灌木，果实富含超氧化歧化酶(SOD)，其具有良好的清除自由基和抗氧化功能；根皮富含皂苷、鞣质、亚麻酸和多种维生素等。果实和根部均可入药，具有增强人体免疫力，祛风湿、强筋骨的功效。刺五加主要分布于黑龙江、吉林、辽宁、河北和山西，近年来，其种植面积逐渐扩大。在刺五加培育和推广中，刺五加果实品质参差不齐，急需优良种质的分子鉴定方法，为高生物活性成分刺五加良种筛选提供科学依据。
微卫星标记(Simple Sequence Repeats Marker，SSR)广泛分布于植物基因组中的不同目的基因上，重复性和稳定性较好。SSR标记已应用在植物遗传图谱构建、多样性分析和分子标记辅助育种等方面。以往，无参考基因组的植物，在开发SSR分子标记时常采用EST序列，需要从大量引物中筛选和验证与性状连锁的SSR标记，工作量大而且具有不确定性和盲目性。目前，未见基于RNA-seq技术开发刺五加目的基因SSR分子标记方面的报道。
发明内容
为了克服现有RNA-seq SSR分子标记筛选方法所具有的工作量大、不确定性和盲目性的不足，本发明提供一种刺五加目的基因SSR分子标记的筛选方法，本发明利用刺五加果实、根、茎和叶的RNA-seq数据开发目的基因SSR分子标记，利用unigene具有基因功能注释且含有SSR位点的特点，克服了现有技术筛选SSR标记的不确定性、盲目性的缺点，有针对性的补充与刺五加品质相关的候选SSR分子标记，进而为刺五加种质资源多样性分析和性状关联分析提供有效标记，也为优良种质资源早期筛选提供分子依据。
本发明的上述目的是通过以下技术方案实现的：
1.分别提取刺五加果实、根、茎和叶的总RNA，等量混合不同组织器官的总RNA；
2.将上述总RNA样品反转录后，进行RNA-seq测序，并利用Trinity软件进行unigene组装；
3.重点利用unigene的基因功能注释，筛选与刺五加皂苷、SOD、维生素、糖、酸、脂肪酸等合成积累相关的unigene，并利用MicroSatellite软件筛选这些unigene上的SSR位点；
4.根据目的基因SSR位点两端互补序列，利用Primer3.0软件设计引物，并合成；
5.以刺五加叶片基因组DNA为模板进行PCR扩增；
6.PCR产物进行电泳检测，筛选符合预期产物大小且条带清晰的目的基因SSR引物。
进一步的，所述步骤3中unigene注释包括Nr、Swiss-Prot、KEGG和COG公共数据库的功能注释。利用KAAS功能将unigene映射到KEGG数据库获得代谢途径信息。根据复杂且数量较多的unigene注释信息，筛选出与目的性状相关且含有SSR位点的unigene(目的基因)，进而更有针对性的设计并开发与目的性状相关的目的基因SSR引物，为刺五加种质的性状关联分析提供候选SSR分子标记。
进一步的，所述步骤6开发目的基因SSR标记的方法包括：(1)根据琼脂糖凝胶电泳结果，初步筛选SSR引物的可用性；(2)采用聚丙烯酰胺凝胶电泳检测，分析目的基因SSR引物扩增条带的准确性和适用性。
本发明与现有技术相比的有益效果是：本发明基于RNA-Seq技术开发刺五加目的基因SSR分子标记，首次有机结合了unigene具有基因功能注释且分布有SSR标记的特点，有针对性的开发与刺五加皂苷、SOD、维生素、糖、酸、脂肪酸等合成积累相关的SSR引物，有效扩增率达到52.3％。所获得的目的基因SSR标记可作为遗传多样性分析、指纹图谱构建和性状关联分析的候选功能标记，补充非常稀少的刺五加SSR标记数量，为优良刺五加种质资源的分子鉴定提供有效标记。克服了现有技术筛选SSR标记的不确定性、盲目性的缺点，对优良刺五加种质资源早期鉴定和加快育种进程具有重要意义。
具体实施方式
下面通过具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从商业途径获得。
实施例
1.提取刺五加果实、根、茎和叶总RNA
以黑龙江省绥棱县的1个刺五加种质SL1为材料。根据上海生工植物RNA提取试剂盒推荐方法提取刺五加不同组织器官的总RNA。各组织器官的总RNA浓度200-400ng/μL，-80℃保存。
2.刺五加果实、根、茎和叶的RNA-seq数据分析
以4个组织器官总RNA等量混合的样品为模板进行反转录，构建转录组文库。将测序得到的raw reads去除接头、N比例大于0.1％和低质量的reads后获得clean reads，利用Trinity软件对clean reads进行拼接得到unigene。将unigene与公共数据库(Nr、Swiss-Prot、KEGG和COG等)中的已知功能基因进行对比。利用KAAS功能将unigene映射到KEGG数据库获得代谢途径信息。
3.筛选与皂苷、SOD、维生素、糖、酸、脂肪酸等合成相关的unigene(且含有SSR位点)
根据unigene注释、代谢通路信息和相关文献报道，筛选与刺五加皂苷、SOD、维生素、糖、酸、脂肪酸等合成相关的unigene。筛选到的目的基因包括与皂苷合成相关的SS(squalene synthase)、HMGR(3-hydroxy-3-methylglutaryl coenzyme A)、SE(squaleneepoxidase)、DXPS(1-deoxy-D-xylulose-5-phosphate synthase)和OSCs(epoxy squalenecyclase)基；与脂类合成相关的acetyl-coA carboxylase、3-ketoacyl-ACP-synthase II、3-ketoacyl-ACP-synthase III、fatty acyl-ACP thioesterase B、fatty acyl-ACPthioesterase A、stearoyl-CoA desaturase、oleate delta-12desaturase、glycerol-3-phosphate acyltransferase、diacylglycerol acyltransferase、mitochondrial trans-2-enoyl-CoA reductase、3-ketoacyl-CoA synthase和phosphatidic acid phosphatase基因；与糖酸合成相关的fructose-bisphosphate aldolase、citrate synthase、malatedehydrogenase、phosphoenolpyruvate carboxylase、alpha-amylase、glucosidase、sucrose synthase、neutral invertase、pectinesterase、polygalacturonase基因；与维生素合成有关的TC(tocopherol cyclase)、GDP-mannose pyrophosphorylase和Myo-inositol oxygenase基因；以及SOD基因。采用MicroSatellite软件检测这些unigene的SSR位点。
4.以刺五加叶片基因组DNA为模板进行PCR扩增
根据改进的天根公司植物基因组DNA提取试剂盒方法提取刺五加叶片基因组DNA，具体方法为：①叶片(20～40mg)充分碾磨后移入离心管，加入400～600μL缓冲液LP1和4～6μL RNase A后漩涡振荡1min；②加入100～200μL缓冲液LP2后漩涡振荡1min；③12000rpm离心5min，转移上清液；④加入1.5倍体积的缓冲液LP3后振荡混匀15s；⑤将所有溶液移入吸附柱CB3中央，放置10min后12000rpm离心2min，弃废液；⑥吸附柱CB3中加入600μL漂洗液PW，13000rpm离心1.5min，弃废液；⑦重复步骤⑥；⑧吸附柱CB3放回收集管，12000rpm离心2min后，将CB3室温放置15min；⑨吸附柱CB3移入新离心管，向吸附膜中央部位悬空滴加36～50μL TE溶液，室温放置5min，12000rpm离心2min；取DNA溶液再次滴入吸附膜中央，室温放置2min，12000rpm离心2min，获得刺五加叶片基因组DNA溶液。
根据步骤3中含有SSR位点的目的基因序列，设计了107对引物，以刺五加叶片基因组DNA为模板进行PCR扩增，反应总体系为20μL：10×PCR buffer 2μL、10mmol/L的dNTP 0.4μL、10μmol/L的上、下游引物各1μL、20ng/μL的模板DNA 2μL、Taq酶0.2μL，ddH2O 13.4μL；反应程序为：94℃预变性4min；94℃变性30s、58℃退火30s、72℃延伸30s、35个循环；72℃延伸10min；4℃保存。
5.电泳检测PCR产物，开发目的基因SSR分子标记
首先利用2.5％琼脂糖凝胶电泳检测107对SSR引物的PCR产物，初步筛选获得56对适用于刺五加的SSR标记。然后，进一步利用8％聚丙烯酰胺凝胶垂直板电泳检测这56个PCR扩增产物(表1)，也获得预期产物大小且清晰的条带。
表1基于RNA-Seq开发的刺五加目的基因SSR引物
Claims (7)
1.一种刺五加目的基因SSR分子标记的筛选方法，其特征在于，以黑龙江省绥棱县的1个刺五加种质SL1为材料；筛选方法包括以下步骤：
步骤1、分别提取刺五加果实、根、茎和叶的总RNA，等量混合不同组织器官的总RNA；
步骤2、将步骤1中的总RNA样品反转录后，进行RNA-seq测序，并利用Trinity软件进行unigene组装；
步骤3、利用unigene具有基因功能注释的特点，筛选与皂苷、SOD、维生素、糖、酸、脂肪酸等合成相关的unigene：且含有SSR位点；根据unigene注释、代谢通路信息筛选与刺五加皂苷、SOD、维生素、糖、酸、脂肪酸等合成相关的unigene；筛选到的目的基因包括与皂苷合成相关的SS(squalene synthase)、HMGR(3-hydroxy-3-methylglutaryl coenzyme A)、SE(squalene epoxidase)、DXPS(1-deoxy-D-xylulose-5-phosphate synthase)和OSCs(epoxy squalene cyclase)基；与脂类合成相关的acetyl-coA carboxylase、3-ketoacyl-ACP-synthase II、3-ketoacyl-ACP-synthase III、fatty acyl-ACP thioesterase B、fatty acyl-ACP thioesterase A、stearoyl-CoA desaturase、oleate delta-12desaturase、glycerol-3-phosphate acyltransferase、diacylglycerolacyltransferase、mitochondrial trans-2-enoyl-CoA reductase、3-ketoacyl-CoAsynthase和phosphatidic acid phosphatase基因；与糖酸合成相关的fructose-bisphosphate aldolase、citrate synthase、malate dehydrogenase、phosphoenolpyruvate carboxylase、alpha-amylase、glucosidase、sucrose synthase、neutral invertase、pectinesterase、polygalacturonase基因；与维生素合成有关的TC(tocopherol cyclase)、GDP-mannose pyrophosphorylase和Myo-inositol oxygenase基因；利用MicroSatellite软件筛选这些unigene上的SSR位点；
步骤4、根据目的基因SSR位点两端互补序列，利用Primer3.0软件设计引物，并合成；
步骤5、以刺五加叶片基因组DNA为模板进行PCR扩增；提取刺五加叶片基因组DNA，具体方法为：①叶片称取20～40mg充分碾磨后移入离心管，加入400～600μL缓冲液LP1和4～6μLRNase A后漩涡振荡1min；②加入100～200μL缓冲液LP2后漩涡振荡1min；③12000rpm离心5min，转移上清液；④加入1.5倍体积的缓冲液LP3后振荡混匀15s；⑤将所有溶液移入吸附柱CB3中央，放置10min后12000rpm离心2min，弃废液；⑥吸附柱CB3中加入600μL漂洗液PW，13000rpm离心1.5min，弃废液；⑦重复步骤⑥；⑧吸附柱CB3放回收集管，12000rpm离心2min后，将CB3室温放置15min；⑨吸附柱CB3移入新离心管，向吸附膜中央部位悬空滴加36～50μL TE溶液，室温放置5min，12000rpm离心2min；取DNA溶液再次滴入吸附膜中央，室温放置2min，12000rpm离心2min，获得刺五加叶片基因组DNA溶液；
步骤6、PCR产物进行电泳检测，筛选符合预期产物大小且条带清晰的目的基因SSR引物；所述刺五加目的基因SSR分子标记筛选方法开发的56组引物组刺五加目的基因SSR引物组合为：
2.根据权利要求1所述的筛选方法，其特征在于，在所述步骤1中，刺五加各组织器官的总RNA浓度200-400ng/μL，-80℃保存。
3.根据权利要求1所述的筛选方法，其特征在于，在所述步骤2中，将测序得到的rawreads去除接头、N比例大于0.1％和低质量的reads后获得clean reads，利用Trinity软件对clean reads进行拼接得到unigene，将unigene与公共数据库中的已知功能基因进行对比，利用KAAS功能将unigene映射到KEGG数据库获得代谢途径信息。
4.根据权利要求1所述的筛选方法，其特征在于，在所述步骤3中，根据复杂且数量较多的unigene注释信息，筛选出与刺五加功能活性成分合成积累相关的unigene，采用MicroSatellite软件检测这些刺五加unigene的SSR位点，进而更有针对性的设计并开发与目的性状相关的目的基因SSR引物，为刺五加种质的性状关联分析提供候选SSR分子标记。
5.根据权利要求1所述的筛选方法，其特征在于，在所述步骤5中，以叶片基因组DNA为模板进行PCR扩增，反应总体系为20μL：10×PCR buffer 2μL、10mmol/L的dNTP 0.4μL、10μmol/L的上、下游引物各1μL、20ng/μL的模板DNA 2μL、Taq酶0.2μL，ddH2O 13.4μL；反应程序为：94℃预变性4min；94℃变性30s、58℃退火30s、72℃延伸30s、35个循环；72℃延伸10min；4℃保存。
6.根据权利要求1所述的筛选方法，其特征在于，在所述步骤6中，开发目的基因SSR标记的方法包括：(1)利用琼脂糖凝胶电泳结果，初步筛选SSR引物的可用性；(2)采用聚丙烯酰胺凝胶电泳检测，分析目的基因SSR引物扩增条带的准确性和适用性。
7.根据权利要求1-6中任一项所述的筛选方法得到的刺五加目的基因SSR分子标记在刺五加种质资源多样性分析和性状关联分析，优良种质资源早期筛选的应用。
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