CN117264966A - MtNAC33基因及其编码蛋白在紫花苜蓿高产抗旱方面的应用 - Google Patents
MtNAC33基因及其编码蛋白在紫花苜蓿高产抗旱方面的应用 Download PDFInfo
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Abstract
本发明涉及一种MtNAC33基因及其编码蛋白在紫花苜蓿高产抗旱方面的应用，属于植物基因工程技术领域，植物抗旱蛋白MtNAC33氨基酸序列如SEQ ID NO.2所示，核苷酸序列如SEQ ID NO.1所示。本发明通过调控紫花苜蓿MtNAC33基因的过表达，能够显著提高拟南芥和紫花苜蓿的抗旱品质，且在紫花苜蓿中出现开花延迟、叶茎比增加的表型，对于牧草及其他作物抗逆性和生物量的遗传改良具有重要的参考意义。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及MtNAC33基因及其编码蛋白在紫花苜蓿高产抗旱方面的应用。
背景技术
紫花苜蓿(Medicago sativa L.)为多年生豆科牧草，紫花苜蓿根系非常发达，具有较强的非生物胁迫抗性。随着紫花苜蓿大面积栽培和集约化生产，对于优质高抗高产的新苜蓿品种需求也日趋急迫。利用基因工程手段进行苜蓿品质改良，能够更快速的培育出优质、高产、高抗逆性的苜蓿品种，推动牧草畜牧业发展。
干旱是限制植物生长、发育和分布的重要环境胁迫之一，其对作物造成的损失在所有非生物胁迫中占居首位，严重影响农业发展。近年来对植物抗旱能力的研究已经逐渐成为全球关注的热点。通过遗传改良将抗旱性较强的牧草施用于旱碱地改良，将实现节约耕地面积和提高旱碱地区畜牧业发展的双重目标。
转录因子位于基因表达调控的上游，往往可以实现多个性状的共同调控，使之成为植物多性状调控的候选靶标之一。NAC蛋白家族是植物特异性的转录因子超家族，在植物生长发育、代谢调控和逆境胁迫等生物过程中均发挥着重要作用。目前，在改良植物抗旱性能的研究中，大多数基因不能够同时满足抗旱和高产的双重需求。因此，利用现代生物学的方法研究紫花苜蓿MtNAC33的基因功能，为提高紫花苜蓿抗旱性能、保证作物产量提供可能。
发明内容
针对背景技术中植物抗旱领域中的技术需求，本发明的目的在于提供一种MtNAC33基因及其编码蛋白在紫花苜蓿高产抗旱方面的应用，解决目前植物抗旱基因资源库不足，不能同时满足改良植株产量和品质分子设计的需要的问题。
本发明是通过如下技术方案来实现的：
本发明提供MtNAC33基因在紫花苜蓿高产抗旱方面的应用，所述MtNAC33基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供所述MtNAC33基因编码的蛋白在紫花苜蓿高产抗旱方面的应用，所述蛋白的氨基酸序列SEQ ID NO.2所示。
本发明还提供一种重组载体pEG100-MtNAC33在紫花苜蓿高产抗旱方面的应用，所述重组载体pEG100-MtNAC33含有MtNAC33基因。
进一步，所述的应用为基于Gateway技术，将MtNAC33基因的全长序列重组整合到表达载体pEG100中；采用农杆菌EHA105介导的遗传转化方法，转入植物中，通过草铵膦抗性筛选，获得过表达MtNAC33基因的抗性再生植株，所述植株不仅具有抗旱性能，而且使植株开花延迟，叶茎比增加，产量提升。
本发明的核心特点和发明理念包括：
1、本发明利用基因工程的手段，通过过量表达技术提高植物高产抗旱蛋白MtNAC33在紫花苜蓿体内的表达水平，能够在短期内获得显著效果；
2、本发明从调控植株耐旱及多种性状的基因着手，在保证作物产量的同时，分析紫花苜蓿基因抗旱胁迫的调控机制，为植物抗旱资源提供更多基因资源。
本发明与现有技术相比的有益效果如下：
1、本发明获得植物高产抗旱蛋白MtNAC33基因是调控苜蓿耐旱品质的关键基因，同时该基因调控苜蓿叶茎比和开花时间，使叶茎比增加，延迟开花，这对于通过分子育种手段获得耐旱植株具有重要意义；
2、本发明中对植物高产抗旱蛋白MtNAC33进行分子调控，能够显著增加耐旱品质。提高紫花苜蓿叶片大小，增加产量。增加紫花苜蓿叶片可溶性蛋白和单糖含量，提高苜蓿品质。对于牧草及其他作物生物量和抗逆性的遗传改良具有重要的参考意义；
3、本发明中所产生的遗传改良植物能够整合到常规育种项目，从而为牧草作物的品种培育提供新的种质资源。
附图说明
图1蒺藜苜蓿中的植物高产抗旱蛋白MtNAC33克隆电泳图；
图2蒺藜苜蓿pEG100-MtNAC33表达载体图；
图3MtNAC33-OE转基因拟南芥和野生型植株中MtNAC33基因定量PCR结果；
图4MtNAC33-OE转基因拟南芥和野生型植株干旱胁迫处理表型图；
图5MtNAC33-OE转基因紫花苜蓿和野生型植株中MtNAC33基因定量PCR结果；
图6MtNAC33-OE转基因紫花苜蓿和野生型植株表型图；
图7MtNAC33-OE转基因紫花苜蓿和野生型植株生物量统计分析图；
图8MtNAC33-OE转基因紫花苜蓿和野生型植株干旱处理表型图；A为离体叶子再24、48、72h后光合作用强度对照图，由植物荧光成像仪检测，B为实验组和野生组干旱处理和不干旱处理15天的生长量；
图9MtNAC33-OE转基因紫花苜蓿和野生型植株单糖品质分析图。A为各组中单糖含量的柱状图，B为各组中淀粉含量的柱状图，C为各组中水溶性碳水化合物含量的柱状图，D为各组中可溶性蛋白占总蛋白的比例的柱状图。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详细说明。下述实施例中所用的材料、试剂和分子标记探针等，如无特殊说明，均可从公司通过商业途径购买。
实施例1：MtNAC33基因的克隆
根据蒺藜苜蓿MtNAC33参考基因组全长序列两侧设计引物：MtNAC33-F和MtNAC33-R，以蒺藜苜蓿的cDNA为模板，进行PCR扩增。
所述引物序列如下：
MtNAC33-F：CCCTTCCGTGCTTCATA
MtNAC33-R：CTCAAATAAATCAGCCATCA
PCR反应体系为：2μL cDNA，5μL 10×Buffer，4μL 2.5mMdNTP，10μM
的正/反向引物各1μL，0.5μL 5U/μLTaq酶和36.5μL ddH2O。在冰上加样后混匀。PCR反应条件为：94℃5min；94℃30sec，56℃45sec；72℃2min，34个循环；72℃10min。
将PCR扩增产物进行1％琼脂糖凝胶电泳检测，得到片段大小为1544bp的片段(图1)，对扩增片段进行凝胶回收(使用Promega凝胶回收试剂盒)，常规测序(北京六合华大基因科技有限公司)，测序结果显示，真实的蒺藜苜蓿基因MtNAC33与参考基因组差异较小，基因全长均为1074bp。如图1所示。
实施例2：过量表达MtNAC33转基因拟南芥的获得
设计过量表达载体中连接入门载体引物：MtNAC33-OE-F和MtNAC33-OE-R，引物末端引入BamHⅠ酶切位点和入门载体pENTRE酶切位点后9个碱基(Infusion接头序列)，以得到的MtNAC33全长序列为模板，用上述引物进行PCR扩增。
所述引物序列如下：
MtNAC33-OE-F：TCCTTCACCCGGGATCCATGGCCGAAACAAAATTGAT
MtNAC33-OE-R：ACCCTTTATCGGGATCCCAGCCATCATGTTTAGTCT
其中，下划线为Infusion接头序列。
回收上述扩增片段。用限制性内切酶BamHⅠ酶切pENTRE载体，并回收。将上述两个片段连接并转化大肠杆菌，得到入门重组载体的阳性克隆并提取质粒。EcoRV核酸内切酶37℃酶切1h，最后通过Gateway技术将目的片段重组到pEG100载体上(图2)。重组反应为：100ng酶切回收片段，50ng pEG100载体质粒，1μL LR酶(Invitrogen，货号11791020)，补齐水至10μL。25℃培养6h。转化大肠杆菌并得到测序正确的阳性重组菌株质粒pEG100-MtNAC33，转化农杆菌EHA105。
将得到的重组农杆菌接种于200mLYEP培养基(含50mg/mL卡纳霉素，50mg/mL利福平)中，过夜培养。收集菌体，室温4000rpm离心10min，用含有10％的蔗糖MS液体培养基稀释OD600为0.9-1.1。使用浸花法转化拟南芥(Col-0)，浸泡5分钟后，用黑色塑料袋遮光24h后正常培养，收取T1代种子，用50μg/L的草甘膦筛选阳性植株。T1代为T0代自交产生的种子及生长的植株，T3代为T2代自交产生的种子及长成的植株。从阳性T3代植株中筛选得到的转基因植株MtNAC33-OE-A和MtNAC33-OE-C作为纯合转基因株系，进行过表达鉴定。
选取阳性植株，用TriZol Reagent试剂盒(Invitrogen公司，货号15596026)提取叶片总RNA，琼脂糖凝胶电泳和核酸分析仪(NanoDrop)检测总RNA的含量和纯度，取1.0μg总RNA做逆转录反应，采用逆转录酶(Promega公司，货号M1701)反转为cDNA，逆转录反应步骤参考该使用说明。以上述cDNA为模板，使用引物MtNAC33-ORF-QF和MtNAC33-ORF-QR进行荧光定量PCR检测，内参基因为拟南芥Actin基因。引物序列如下：
AtActin-F：GGTAACATTGTGCTCAGTGGTGG，
AtActin-R：AACGACCTTAATCTTCATGCTGC；
MtNAC33-ORF-QF：AAAGACTGGGATAGCGAAGA，
MtNAC33-ORF-QR：CCTGGAGCTGAAGGGTGT。
实时荧光定量PCR反应体系为20μL，其中正/反向引物各1μL，cDNA模板2μL，SYBRGreen qRT MasterMix(购自宝生物工程有限公司)10μL，ddH2O补足至20μL。实时荧光定量PCR仪使用Roche480，使用两步法反应。检测结果表明，于野生型Col-0相比，转基因植株MtNAC33-OE-A和MtNAC33-OE-C中MtNAC33的表达量均显著上升(图3)。
实施例3：过量表达MtNAC33转基因拟南芥抗旱能力检测
将经实施例2的荧光定量PCR鉴定分析结果MtNAC33表达上调的T3代转基因植株MtNAC33-OE-A和MtNAC33-OE-C作为实验株系，与野生型拟南芥Col-0一起进行干旱测试。相同条件下培养1个月左右，分别进行正常浇水(图4上)与不浇水处理(图4下)，10天之后，过表达株系较野生型表现出明显的耐旱能力(图4)。
综上，通过上述转基因，将MtNAC33过量表达转化到植株中可显著增强植株抗旱能力。
实施例4：过量表达MtNAC33转基因紫花苜蓿植物的获得
将实施例2中得到的阳性重组菌株质粒pEG100-MtNAC33转化的农杆菌EHA105接种于50mLYEP培养基(含50mg/mL卡纳霉素，50mg/mL利福平)中，过夜培养摇至OD600为0.6-0.8。收集菌体，室温4000rpm离心10min，用转化液(含有200μMAS的SH3a培养液)重悬菌体，孵育2h，最终稀释至OD600为0.2-0.3。取紫花苜蓿新鲜幼嫩叶片用0.5％次氯酸钠消毒，将消毒后的叶片剪至1cm2左右大小，倒入制备好的菌液，超声15min，倒掉菌液，将叶片摆放于共培养培养基(SH3a+100μMAS)上，黑暗培养至少24h。将共培养后的转化叶片转移至筛选培养基上，黑暗培养，诱导愈伤。将诱导的愈伤转移至分化培养基，约1-2周出现绿色芽点后转至SH9分化培养基，获得抗性苗。经阳性鉴定后进行过量表达鉴定。
取转基因阳性植株嫩叶组织，按照实例2中的方法进行植株总RNA提取和逆转录，得到紫花苜蓿cDNA。以上述cDNA为模板，使用引物MtNAC33-ORF-QF和MtNAC33-ORF-QR进行荧光定量PCR检测，内参基因为紫花苜蓿Actin基因。引物序列如下：
MsActin-F：CCCACTGGATGTCTGTAGGT；
MsActin-R：AGAATTAAGTAGCAGCGCAAA。
实时荧光定量PCR反应体系与方法如实例2所述。检测结果表明，与野生型紫花苜蓿相比，转基因植株中MtNAC33的表达量均显著上升，(图5显示部分转基因紫花苜蓿定量分析结果(MtNAC33-OE-6/10/17/1/11/15))。
实施例5：过量表达MtNAC33转基因紫花苜蓿的表型
将实施例4中的荧光定量PCR鉴定分析结果MtNAC33表达上调的转基因植株分别按照其表达水平与相对应的表型进行分组。结果显示，在转基因过程中，约50％的转基因株系发生MtNAC33表达量为野生型的2-5倍的情况(MtNAC33-OE-6/10/17)，现蕾期分析结果表明，转基因植株叶片变大、开花延迟、叶茎比增加以及生物量(图6和图7)，这一性状对植株生产具有重要意义。
实施例6：过量表达MtNAC33转基因紫花苜蓿抗旱能力显著提高
将实施例5中MtNAC33表达上调且生物量显著提高的转基因植株(MtNAC33-OE-6/17)与对照植株中苜1号进行叶片干旱测试。过量表达MtNAC33转基因紫花苜蓿叶片为实验组，对照组为野生植株，结果显示，对照组和实验组叶片在离体放置48h时，实验组的光合作用显现强于对照组，72h时依然保持一定的光合能力(图8中的A)(图片颜色越深代表光合作用能力越强)，对照组叶片已经枯萎，丧失了光合作用的能力。此外，植株干旱处理试验表明，停水15天后，转基因植株与对照相比表现出较强的耐旱性，图8中的B可以看出，实验组叶片仍然保持绿色，对照组则出现黄叶。因此，利用MtNAC33在紫花苜蓿中过量表达能够显著提高紫花苜蓿耐旱能力。
实施例7：过量表达MtNAC33转基因紫花苜蓿干物质品质检测
将实施例5中的现蕾期材料使用近红外扫描检测(Dairy One数据库)对紫花苜蓿的基础营养指标进行测定，其中过表达MtNAC33转基因紫花苜蓿叶片中的单糖、淀粉、水溶性碳水化合物、可溶性蛋白占总蛋白的比例均有增加(图9)。MtNAC33基因(SEQ ID NO.1)
ATGGCCGAAACAAAATTGATGATTCCAGGGTTTCGTTTTCACCCCACTGAT
GTTGAGCTGGTAATGTATTTTCTCAAGAGGAAGATTTTGGGTAGAAAATTC
CCTTTTAATGTCATTGATGAACTTGACATTTACAAGTATGCTCCATGGGATCT
ACCAGAAAAATCTTTGCTCAAGAGTGGTGATTTGCAATGGTACTTCTTTAC
CCCTGTCGGAAAGAAATATTGCACGGGAGGGAGGATGAATCGGGCAACAG
AAGTAGGCTACTGGAAGACTACAGGGAAGGATAGGTCGATTGAACATAGG
AATCAAGTGGTGGGGATGATAAGAACCCTGGTGTTTCACACTGGCAAAGC
TCCTAAAGGAGACCGAACTGATTGGGTTATGCATGAATACAGACTTGAAAA
CAAAGACCTAGCTGACAATGGTGTTCCACAGAACTCCTATGTGATTTGTAG
GGTATTTCAAAAGGAAGGTCCTGGTCCCAGGAATGGTGCACAGTATGGAA
AACCATTTAATGAGAAAGACTGGGATAGCGAAGAGGAAATTGATTATGTAC
AAGCTGTCCCTGTTGCTGCTGTGTCTGCACCAGCTCTCATTCTACCTAGCTC
AAGCCATATTTCTGAAGAAAATGATATGCATACCTCTGCAAGGGGATGCAC
TGGACAGACCTCTTTATCAGGTCTATCAAGATTGATGCCCTCTGGCACGAC
ACACCCTTCAGCTCCAGGCAATCAAGCTGATGATGACATTTTATCCATGCTT
GCTATCTTTGACGATGAAAATGCATTGGCTGGGAATGAAAACAATGGATCT
GAGAAGGTCGATAATCCTGGTCAGGCAAACAATGCTGAAGATGTACCTTAT
TTAATTTCAAATGAGATTTTTGAGGACTTGGGAGATCTCAACAGCTTGGTT
GGATTAGATGAAGGAGGCGGTTTTTCCTATGGCCAAAAAGATGAGTACGA
AAAGCTTTCTACTGGCAACGTCTCTTTGTTTTGCAACCCCCCTGATTTCTTT
GAGTTGCTTGACCTAGAGGTGCCATTATCTTGGCAGACTAAACATGATGGCTGA。
MtNAC33蛋白(SEQ ID NO.2)
MAETKLMIPGFRFHPTDVELVMYFLKRKILGRKFPFNVIDELDIYKYAPWDLPEKSL
LKSGDLQWYFFTPVGKKYCTGGRMNRATEVGYWKTTGKDRSIEHRNQVVGMIRTLVF
HTGKAPKGDRTDWVMHEYRLENKDLADNGVPQNSYVICRVFQKEGPGPRNGAQYGKP
FNEKDWDSEEEIDYVQAVPVAAVSAPALILPSSSHISEENDMHTSARGCTGQTSLSG
LSRLMPSGTTHPSAPGNQADDDILSMLAIFDDENALAGNENNGSEKVDNPGQANNAE
DVPYLIPNEIFEDLGDLNSLVGLDEGGGFSYGQKDEYERLSTGNVSLFCNPPDFFELLDLEVPLSWQTKHDG*。
Claims (4)
1.MtNAC33基因在紫花苜蓿高产抗旱方面的应用，其特征在于，所述MtNAC33基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述MtNAC33基因编码的蛋白在紫花苜蓿高产抗旱方面的应用，其特征在于，所述蛋白的氨基酸序列SEQ ID NO.2所示。
3.一种重组载体pEG100-MtNAC33在紫花苜蓿高产抗旱方面的应用，其特征在于，所述重组载体pEG100-MtNAC33含有权利要1所述的MtNAC33基因。
4.根据权利要求1所述的应用，其特征在于，所述的应用为基于Gateway技术，将MtNAC33基因的全长序列重组整合到表达载体pEG100中；采用农杆菌EHA105介导的遗传转化方法，转入植物中，通过草铵膦抗性筛选，获得过表达MtNAC33基因的抗性再生植株，所述植株不仅具有抗旱性能，而且使植株开花延迟，叶茎比增加，产量提升。
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