CN116479012A - 一个控制大蒜假茎和叶片长度基因AsaPSL1及其应用 - Google Patents
一个控制大蒜假茎和叶片长度基因AsaPSL1及其应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域，涉及一种控制大蒜假茎和叶片长度基因AsaPSL1及其应用；根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段；构建目的基因过量表达载体；将AsaPSL1的过表达载体转化农杆菌GV3101；利用无菌培养的大蒜根诱导形成愈伤组织，再用含有AsaPSL1过表达载体的农杆菌侵染大蒜愈伤组织获得转基因大蒜；通过卡那霉素抗性筛选，获得抗性苗，再使用激光共聚焦显微镜观察EGFP荧光获得阳性转基因苗，表型观察显示AsaPSL1有促进大蒜假茎和叶伸长生长的能力，能够正向调节大蒜的假茎和叶伸长生长，为筛选优势茎叶伸长生长基因提供了新的选择，在葱属基因工程领域有重要的价值。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一种控制大蒜假茎和叶片长度基因AsaPSL1及其应用。
背景技术
大蒜属于石蒜科(Amaryllidaceae)葱属(Allium)，长期以来作为风味蔬菜和天然药用材料被广泛应用。大蒜的独特风味和药用特性来自于活性含硫化合物，主要是蒜氨酸。蒜氨酸在蒜氨酸酶的催化生成大蒜素，具有抗菌消炎、降血脂、预防心脑血管疾病和抗肿瘤等作用。大蒜中蒜氨酸的合成主要是在叶片进行，从半胱氨酸起始合成谷胱甘肽进一步经由几步反应合成蒜氨酸。青蒜是指蒜头未成熟的嫩茎和叶。青蒜在颜色、硬度、香味上都与成熟的大蒜或葱有所不同。其呈浅绿色或淡黄绿色，茎细而嫩，口感略带嫩笋的口感。青蒜是中国烹饪中常用的蔬菜之一，味道清新爽口，营养丰富，具有一定的药用价值。青蒜含有多种维生素和矿物质，如维生素C、硒、锰、铁等，还含有丰富的挥发油和硫化物，对身体健康有一定的功效。青蒜可用于烹饪多种菜肴，如青蒜炒鸡蛋、鱼香青蒜虾仁、青蒜烤鱼等。此外，青蒜也可入药，具有清热解毒、发汗解表、止咳化痰等功效。目前大蒜遗传转化体系不成熟，鲜有报道大蒜基因工程领域内容，严重阻碍了大蒜分子育种研究。
通过培育茎叶生长良好的大蒜新品种，提高青蒜产量是大蒜品种改良亟需解决的重要问题。
因此，提供一种控制大蒜假茎和叶片长度基因AsaPSL1及其应用，有助于了解和深入研究大蒜的生长机理，为后续遗传育种，创制假茎和叶片长，青蒜产量高的新品种，打下良好基础。
发明内容
本发明的目的在于解决现有技术中的不足，提供一种控制大蒜假茎和叶片长度基因AsaPSL1及其应用，通过农杆菌介导的遗传转化体系获得转基因大蒜，验证目的基因功能，以期促进大蒜分子育种技术的发展，为培育假茎和叶片长、青蒜产量高新品种提供基因资源，同时为探索大蒜假茎和叶片伸长生长的分子机理奠定基础。
为了达到上述目的，本发明是通过以下技术方案实现的：
第一方面，本发明提供一种控制大蒜假茎和叶片长度基因AsaPSL1，所述基因AsaPSL1的核苷酸序列如SEQ ID No.1所示。
所述AsaPSL1编码的氨基酸序列如SEQ ID No.2所示。
第二方面，本发明提供所述的控制大蒜假茎和叶片长度基因AsaPSL1在调控大蒜假茎及叶伸长生长中的应用。
所述应用的方法如下：
步骤(1)：根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段；
步骤(2)：构建目的基因过表达载体；
步骤(3)：利用农杆菌介导的方法进行转基因操作；
步骤(4)：获得转基因大蒜。
优选的，步骤(1)根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段，具体步骤如下：使用RNA提取试剂盒提取大蒜叶片RNA，用M-MLV first strand cDNASynthesis Kit反转录合成cDNA；PCR扩增目的基因，得到PCR产物。
优选的，所述PCR扩增目的基因，扩增体系为：25μl 2X ApexHF FS PCR MasterMix,1μl AsaPSL1-F,1μl AsaPSL1-R,2μl cDNA，补充无菌水至50μl；PCR反应程序为：95℃3min；98℃ 10sec,56℃ 5sec，72℃ 90sec,30个循环；72℃ 3min；
引物如下：
| 序列名称 | 序列 | 序列编号 |
| AsaPSL1-F | ATGGTCTCTGAATATATCAACTCC | SEQ ID No.3 |
| AsaPSL1-R | TCATATTTTAATAACATCTGGATCTAGCT | SEQ ID No.4 |
。
优选的，步骤(2)构建目的基因过表达载体，具体步骤如下：将基因片段连接到过表达载体PBI121-EGFP，获得35S启动子驱动的AsaPSL1过量表达框，得到过表达载体PBI121-EGFP-AsaPSL1。
优选的，载体连接用的引物如下：
| 序列名称 | 序列 | 序列编号 |
| pbi121-egfp-AsaPSL1-f | tggagagaacacgctcgagATGGTCTCTGAATATATCAAC | SEQ ID No.5 |
| pbi121-egfp-AsaPSL1-r | gcggcagcagccggatccTCATATTTTAATAACATCTGG | SEQ ID No.6 |
。
优选的，步骤(3)利用农杆菌介导的方法进行转基因操作，具体步骤如下：采用冻融法，将所构建的过表达载体转入农杆菌GV3101中，通过农杆菌介导的遗传转化转入大蒜愈伤中；转化步骤如下：用于遗传转化的大蒜愈伤组织培养在温度为22℃，黑暗培养；含有目的表达载体的农杆菌在OD＝0.6时侵染愈伤，侵染后的愈伤组织，在共培养基黑暗培养3天，共培养后的愈伤用无菌水清洗3次后转移至抗性筛选培养基，22℃培养，光照为：白天16h/黑夜8h，光照强度为2500Lux；三周更换一次培养，直到分化出不定芽；将不定芽切下转移入生根培养基MS+100mg/l卡那霉素；待分化苗长到10cm以上，根系发育良好后转移到无菌土壤中培养。
本发明具有以下有益效果：本发明以二水早大蒜为材料，克隆出AsaPSL1基因，基于其过表达的表型鉴定表明，其促进大蒜假茎和叶伸长生长的能力，说明AsaPSL1能够正向调节大蒜的假茎和叶伸长生长，为筛选优势茎叶伸长生长基因提供了新的选择，在葱属基因工程领域有重要的价值。
附图说明
图1：AsaPSL1基因克隆电泳图；
图2：过表达载体示意图，35S启动子驱动目的基因表达，下游连接EGFP绿色荧光蛋白；
图3：转基因大蒜激光共聚焦显微镜观察图；
图4：转基因大蒜和野生型表型图(转基因大蒜假茎更高，叶片更长)；
图5：转基因大蒜和野生型大蒜叶片长度统计箱线图；
图6：转基因大蒜和非转基因大蒜假茎高度统计箱线图；
图7：转基因大蒜和非转基因大蒜叶片表型图。
具体实施方式
下面结合实施例对本发明作进一步的说明，但并不作为对本发明限制的依据。
1、克隆AsaPSL1基因
根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段。根据大蒜基因组序列，设计AsaPSL1基因扩增引物，扩增其编码序列(coding sequence)。使用Omega公司的RNA提取试剂盒提取大蒜叶片RNA，用M-MLV first strand cDNA Synthesis Kit(OMEGA)反转录合成cDNA。大蒜叶片来源为市售大蒜“二水早”。PCR扩增目的基因，扩增体系为：25μl 2XApexHF FS PCR Master Mix,1μl AsaPSL1-F(10μM),1μlAsaPSL1-R,2μl cDNA，补充无菌水至50μl；PCR反应程序为：95℃3min；98℃10sec,56℃5sec，72℃90sec,30个循环；72℃3min,得到PCR产物。使用omega胶回收试剂盒回收PCR产物用于测序验证及后续试验。
AsaPSL1-F(SEQ ID No.3)：ATGGTCTCTGAATATATCAACTCC；
AsaPSL1-R(SEQ ID No.4)：TCATATTTTAATAACATCTGGATCTAGCT。
2、过表达载体构建
将AsaPSL1基因的胶回收产物使用引物pbi121-egfp-AsaPSL1-f和
pbi121-egfp-AsaPSL1-r进行PCR扩增产生具有载体同源臂的片段，扩增反应条件如上一步；再用诺唯赞ClonExpress II同源重组克隆试剂盒，将AsaPSL1基因连接到过表达载体PBI121-EGFP，获得35S启动子驱动的AsaPSL1过量表达框。载体连接用到的引物如下：pbi121-egfp-AsaPSL1-f(SEQ ID No.5):
tggagagaacacgctcgagATGGTCTCTGAATATATCAAC
pbi121-egfp-AsaPSL1-r(SEQ ID No.6):
gcggcagcagccggatccTCATATTTTAATAACATCTGG
3、AsaPSL1基因的遗传转化
利用农杆菌介导的方法进行转基因操作，将AsaPSL1的过表达载体PBI121-EGFP-AsaPSL1转化农杆菌GV3101；利用无菌培养的大蒜根诱导形成愈伤组织，再用含有AsaPSL1过表达载体的农杆菌侵染大蒜愈伤组织获得转基因大蒜。
采用冻融法，将所构建的过表达载体(PBI121-EGFP-AsaPSL1)转入农杆菌GV3101中，通过农杆菌介导的遗传转化转入大蒜愈伤中，转化步骤如下：用于遗传转化的大蒜愈伤组织培养在温度为22℃，黑暗培养。含有目的表达载体的农杆菌在OD＝0.6时侵染愈伤，侵染后的愈伤组织，在共培养基(MS固体培养基)黑暗培养3天，共培养后的愈伤用无菌水清洗3次后转移至抗性筛选培养基(MS+100mg/l卡纳霉素+1mg/l 2,4-D+0.1mg/l IAA)，22℃培养，光照为16/8h(白天/黑夜)，光照强度为2500Lux。三周更换一次培养，直到分化出不定芽。将不定芽切下转移入生根培养基MS+100mg/l卡那霉素。待分化苗长到10cm以上，根系发育良好后转移到无菌土壤中培养。
4、转基因苗验证，获得阳性转基因大蒜
通过激光共聚焦显微镜检测获得的转基因大蒜瓣和叶是否有绿色荧光确定为转基因大蒜。将蒜瓣顶端切下薄片或将叶片切成薄片，用488nm的激发光激发绿色荧光蛋白，然后检测509nm的发射波，在显微镜下可以看到绿色荧光，证明为转基因大蒜。
5、转基因大蒜F1代表型观察统计
将获得的转基因大蒜，种植于室外土培盆中。2个月后统计大蒜叶片长度，4个月后统计假茎高度，统计数据如表1，如图5-6箱线图所示转基因的大蒜假茎和叶与非转基因大蒜具有显著差异，AsaPSL1过量表达的大蒜叶更长且假茎更高。如图4和图7所示，转基因大蒜相比于非转基因大蒜假茎更高、叶片更长。确定AsaPSL1基因能够促进大蒜假茎和叶片的伸长。
表1大蒜叶片和假茎表型统计数据
SEQ ID NO.1：
ATGGTCTCTGAATATATCAACTCCAAGCATGTGTGTGTAATTGGTGCTGGTCCATCA
GGCTTAGTGTCAGCTAGAGAACTAAGAAAAGAAGGACACAGTGTCGTGGTACTAG
AGCAAAATTTTGATGTTGGTGGGCAATGGCTATATGAACCTAACATAGAAAAAGAA
GATACATTGGGTAGAAAAACACCTTTAAAAGTACACAGTAGCGTGTATGCTTCACT
TAGGCTAAATTCTCCGAGGGAGATCATGGGATATACCGACTTTCCATTTCACTCGAA
AAAAGGAAGAGACACGAGAAGGTTTCCAGGTCACGCTGAACTTTTATCTTATCTTA
GAGATTTCTGTCTGCGGTTTAGACTGAGAGAGATGATAAGGTTCAATACTAGAGTT
GAGTATGTAGGCATGACGAATACCAGGGATTTCAGGAATTTAAAATGGATTGTGAG
ATCTAAGGAGCTGGAAAGGGGTGTGGTTAAGGAAGAAATTTTTGATGCAGTGGTC
GTGGCCAATGGACATTACTCGAAACCTAGACTGCCAAGCATCAAAGGAATGGATA
CATGGACAAGAAAACAGATTCATAGCCATGTATACCGGGTTCCCGGCCCCTTTCGA
AATGAGGTAGTGGTAGTTATTGGCAATTCTATGAGTGGCCAAGACATATCCTTGGA
GTTGGTGGGAGTAGCAAAGGAAGTACACATCAGCACCAAATCCCTTGACATCTCG
GAAGGCCTATCGAAAATAATCGACAAATATCAAAACTTACACATACATCTACAGGT
AGACTGCCTATATGGAGACGGAAAGGTTTTGTTTGTTGATGGTACTTGGGTCATTG
CAAATTCTATAATTTATTGCACTGGGTACTCTTATTCATTCTCATTTCTTGACACCAA
AGGGATAGTTTCAATCGACGACGATAGAGTTGGACCTTTATATGAACACACTTTTCC
ACCATCACTTGCCCCATCTCTGTCTTTTGTAGGCATTCCAAGAAAGCTCATAGGATT
TCCATTTTTTGAATCACAAGCTATATGGATCGCACAAGTATTATCAGATAAAAGAAA
ACTACCATCATGGGATACAATGATGAGAGCGATTACAGAATTCTATCGGTTAAAAGA
TGATGCAGGCATACCGAAACACAATACACATGATCTTGCTGATTTTGAGTACTGTGA
TAGATATGGAGACAACTGCGGATTCCCACACTTGGAAGAATGGAGAAAAGAACTT
TGTTTATCTGCTCTAAAAAATGCAGAAATGAACTTAGAAAGTTTTCGAGATATATTT
GAAGACCTTGAACTACTTCAAGAAGCGCACAAAAGCCCTCATTTCATGCAGCTAGATCCAGATGTTATTAAAATATGA。
SEQ ID NO.2：
MVSEYINSKHVCVIGAGPSGLVSARELRKEGHSVVVLEQNFDVGGQWLYEPNIEKED
TLGRKTPLKVHSSVYASLRLNSPREIMGYTDFPFHSKKGRDTRRFPGHAELLSYLRDF
CLRFRLREMIRFNTRVEYVGMTNTRDFRNLKWIVRSKELERGVVKEEIFDAVVVANG
HYSKPRLPSIKGMDTWTRKQIHSHVYRVPGPFRNEVVVVIGNSMSGQDISLELVGVAK
EVHISTKSLDISEGLSKIIDKYQNLHIHLQVDCLYGDGKVLFVDGTWVIANSIIYCTGY
SYSFSFLDTKGIVSIDDDRVGPLYEHTFPPSLAPSLSFVGIPRKLIGFPFFESQAIWIAQVL
SDKRKLPSWDTMMRAITEFYRLKDDAGIPKHNTHDLADFEYCDRYGDNCGFPHLEE
WRKELCLSALKNAEMNLESFRDIFEDLELLQEAHKSPHFMQLDPDVIKI。
Claims (9)
1.一种控制大蒜假茎和叶片长度基因AsaPSL1，其特征在于，所述基因AsaPSL1的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的控制大蒜假茎和叶片长度基因AsaPSL1，其特征在于，所述AsaPSL1编码的氨基酸序列如SEQ ID No.2所示。
3.一种如权利要求1或2所述的控制大蒜假茎和叶片长度基因AsaPSL1在调控大蒜假茎及叶伸长生长中的应用。
4.根据权利要求3所述的应用，其特征在于，所述应用的方法如下：
步骤(1)：根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段；
步骤(2)：构建目的基因过表达载体；
步骤(3)：利用农杆菌介导的方法进行转基因操作；
步骤(4)：获得转基因大蒜。
5.根据权利要求3所述的应用，其特征在于，步骤(1)根据AsaPSL1序列设计引物，从大蒜叶片中获得目的基因片段，具体步骤如下：使用RNA提取试剂盒提取大蒜叶片RNA，用M-MLV first strand cDNA Synthesis Kit反转录合成cDNA；PCR扩增目的基因，得到PCR产物。
6.根据权利要求5所述的应用，其特征在于，所述PCR扩增目的基因，扩增体系为：25μl2X ApexHF FS PCR Master Mix,1μl AsaPSL1-F,1μl AsaPSL1-R,2μl cDNA，补充无菌水至50μl；PCR反应程序为：95℃3min；98℃10sec,56℃5sec，72℃90sec,30个循环；72℃3min；
引物如下：
。
7.根据权利要求3所述的应用，其特征在于，步骤(2)构建目的基因过表达载体，具体步骤如下：将基因片段连接到过表达载体PBI121-EGFP，获得35S启动子驱动的AsaPSL1过量表达框，得到过表达载体PBI121-EGFP-AsaPSL1。
8.根据权利要求7所述的应用，其特征在于，载体连接用的引物如下：
。
9.根据权利要求3所述的应用，其特征在于，步骤(3)利用农杆菌介导的方法进行转基因操作，具体步骤如下：采用冻融法，将所构建的过表达载体转入农杆菌GV3101中，通过农杆菌介导的遗传转化转入大蒜愈伤中；转化步骤如下：用于遗传转化的大蒜愈伤组织培养在温度为22℃，黑暗培养；含有目的表达载体的农杆菌在OD＝0.6时侵染愈伤组织，侵染后的愈伤组织，在共培养基黑暗培养3天，共培养后的愈伤用无菌水清洗3次后转移至抗性筛选培养基，22℃培养，光照为：白天16h/黑夜8h，光照强度为2500Lux；三周更换一次培养，直到分化出不定芽；将不定芽切下转移入生根培养基MS+100mg/l卡那霉素；待分化苗长到10cm以上，根系发育良好后转移到无菌土壤中培养。
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