CN117363623A - 甘蔗木质素相关基因ScDIR11及其在抗黑穗病甘蔗育种中的应用 - Google Patents
甘蔗木质素相关基因ScDIR11及其在抗黑穗病甘蔗育种中的应用 Download PDFInfo
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Abstract
本发明公开了甘蔗木质素相关基因ScDIR11及其在抗黑穗病甘蔗育种中的应用。发明人通过研究甘蔗木质素合成相关基因ScDIR11发现，过表达甘蔗本身的ScDIR11基因，可导致转基因甘蔗的木质素含量提高，甘蔗鞭黑粉菌侵染转基因甘蔗显示其黑穗病发病率显著降低，且转基因植株内的甘蔗鞭黑粉菌含量下降。据此，发明人建立了一种获得高抗黑穗病甘蔗的方法，将含有甘蔗ScDIR11基因的质粒通过根癌农杆菌介导法遗传转化至甘蔗中，即可获得高抗黑穗病甘蔗材料。因此，本发明通过克隆基因ScDIR11，有利于阐明甘蔗对甘蔗鞭黑粉菌的抗病机制，并通过基因工程手段应用于高抗病性甘蔗的定向分子育种，为利用甘蔗ScDIR11基因防治甘蔗黑穗病提供了技术支持。
Description
技术领域
本发明属于甘蔗基因育种技术领域，尤其涉及一种甘蔗木质素相关基因ScDIR11及其在抗黑穗病甘蔗育种中的应用。
背景技术
甘蔗(Saccharum officinarum L.)是一年或多年生的草本植物，也是世界主要的糖料和生物能源作物。目前有上百个国家种植甘蔗，其中，巴西的种植面积最大，其次是印度，第三是中国。甘蔗生产采用茎节作为种苗，但甘蔗育种主要采用有性杂交的方法。现代甘蔗为异源高倍性非整倍体作物，遗传背景极其复杂，将众多的优良性状聚合到一个甘蔗品种上的难度很大。但由甘蔗鞭黑粉菌入侵甘蔗引起的甘蔗黑穗病几乎在所有种植甘蔗的国家和地区被发现。我国甘蔗大部分种植于旱地，有利于甘蔗鞭黑粉菌冬孢子越冬和传播，加之中国蔗区主栽品种较为单一，农艺性状好、适应性广、产量高且糖分高的甘蔗品种很多都感黑穗病，因此甘蔗黑穗病已经成为甘蔗生产中最严重的真菌病害。
甘蔗鞭黑粉菌是引起甘蔗黑穗病的病原真菌，属于担子菌亚门黑粉菌目，其双核菌丝入侵甘蔗组织后，不断吸取寄主的营养物质而生长，侵染后期会在甘蔗末梢长出一条由甘蔗鞭黑粉菌冬孢子包裹甘蔗组织形成的“黑色鞭状”结构，其中包含成熟的暗褐色冬孢子及植物残余组织，罹病植株完全丧失经济价值。新植蔗的黑穗病发病率在2％-5％之间，一年宿根的发病率提高到8％-15％，两年宿根的发病率则高达20％以上，导致甘蔗平均宿根年限仅为两年，对我国蔗糖业的发展构成了严重的威胁。
培育抗病新品种是防治甘蔗黑穗病最为经济有效的措施。甘蔗是无性繁殖作物，且具有极其复杂的遗传背景，通过杂交育种实现甘蔗品种的改良会受到多种因素的制约。因此，通过基因工程的手段选育优良的甘蔗品种，可以极大的缩短育种周期，避免因甘蔗基因组重复序列过多而带来的杂交育种困难。迄今为止，尚没有稳定有效的提高甘蔗黑穗病抗性的基因工程技术。
关于甘蔗DIR基因(ScDIR)的功能尚未被系统阐明，也没有利用甘蔗DIR基因进行甘蔗抗病育种的相关报道。
发明内容
本发明要解决的技术问题是提供一种甘蔗木质素相关基因ScDIR11及其在抗黑穗病甘蔗育种中的应用。
为解决上述技术问题，本发明采用以下技术方案：
甘蔗ScDIR11基因，具有序列表SEQ.ID.NO.1的碱基序列。
含有上述甘蔗ScDIR11基因的遗传转化质粒。
上述遗传转化质粒的构建方法，以单子叶植物的强启动子Ubi作为遗传转化载体的启动子，将Ubi片段和pCAMBIA3300-NOS载体用限制性内切酶Bam HI和Hind III连接得到质粒pCAMBIA3300-Ubi-NOS，将PCR扩增的甘蔗ScDIR11基因与pCAMBIA3300-Ubi-NOS质粒连接，即得pCAMBIA3300-Ubi-ScDIR11-NOS。
甘蔗ScDIR11基因在调控甘蔗木质素产生中的应用。
甘蔗ScDIR11基因在防治甘蔗黑穗病中的应用。
甘蔗ScDIR11基因在抗黑穗病甘蔗育种中的应用。
获得高抗黑穗病甘蔗的方法，将上述遗传转化质粒通过根癌农杆菌介导法遗传转化至甘蔗中。
针对目前甘蔗黑穗病防治存在的问题，发明人通过研究甘蔗ScDIR11基因发现，过表达甘蔗本身的ScDIR11基因，可导致转基因甘蔗的木质素含量提高，甘蔗鞭黑粉菌侵染转基因甘蔗显示其黑穗病发病率显著降低，且转基因植株内的甘蔗鞭黑粉菌含量降低。据此，发明人建立了一种获得高抗黑穗病甘蔗的方法，将含有甘蔗ScDIR11基因的质粒通过根癌农杆菌介导法遗传转化至甘蔗中，即可获得高抗黑穗病甘蔗材料，从而提高甘蔗对甘蔗鞭黑粉菌的抗性。因此，本发明通过克隆甘蔗中的木质素合成基因ScDIR11，有利于阐明甘蔗对甘蔗鞭黑粉菌的抗病机制，并通过基因工程手段应用于高抗病性甘蔗的定向分子育种，为利用甘蔗ScDIR11基因防治甘蔗黑穗病提供了技术支持。
附图说明
图1是ScDIR11基因的PCR扩增产物电泳图。
图2是遗传转化载体pCAMBIA3300-Ubi-ScDIR11-NOS的构建示意图。
图3是酶切验证pCAMBIA3300-Ubi-NOS载体电泳图，图中：泳道1中小片段为Ubi片段，大片段为pCAMBIA3300-NOS片段。
图4是酶切验证pCAMBIA3300-Ubi-ScDIR11-NOS载体电泳图，图中：泳道1中小片段为ScDIR11片段，大片段为pCAMBIA3300-Ubi-NOS片段。
图5是PCR验证转入pCAMBIA3300-Ubi-NOS空质粒的转基因甘蔗株系中的Bar基因电泳图，图中：M为Marker，1-42分别为转基因甘蔗株系样品。
图6是PCR验证转入pCAMBIA3300-Ubi-ScDIR11-NOS质粒的转基因甘蔗电泳图，图中：A和B验证Bar基因，C和D验证ScDIR11-NOS片段，M为Marker，1-39分别为转基因甘蔗株系样品。
图7是qRT-PCR验证转入pCAMBIA3300-Ubi-ScDIR11-NOS质粒的转基因甘蔗基因的相对表达量热图(Heml 1.0.3.7软件)，图中：WT为非转基因甘蔗，line1-36分别为转基因甘蔗株系样品。热图的色标表示表达量，蓝色表示转录物丰度水平低，而红色表示转录物丰度水平高。
图8是甘蔗的木质素含量分析图，图中：纵坐标表示每毫克甘蔗芽内的木质素含量，横坐标表示甘蔗植株，WT为非转基因甘蔗，35S::00表示转入pCAMBIA3300-Ubi-NOS载体的甘蔗株系，35S::ScDIR11表示转入ScDIR11基因的转基因甘蔗株系。
图9是甘蔗鞭黑粉菌接种甘蔗的黑穗病发病率分析图，图中：纵坐标表示甘蔗的黑穗病发病率，横坐标表示各甘蔗株系的发病统计时间，n为甘蔗株树，WT为非转基因甘蔗，35S::00表示转入pCAMBIA3300-Ubi-NOS载体的甘蔗株系，35S::ScDIR11表示转入ScDIR11基因的转基因甘蔗株系。
图10是甘蔗的黑穗菌含量分析图，图中：纵坐标表示甘蔗鞭黑粉菌含量，即用每微克甘蔗DNA中特异片段的拷贝数表示，以10为底作基因拷贝数的对数；横坐标表示甘蔗株系，WT为非转基因甘蔗，35S::00表示转入pCAMBIA3300-Ubi-NOS载体的甘蔗株系，35S::ScDIR11表示转入ScDIR11基因的转基因甘蔗株系。
具体实施方式
实施例1甘蔗ScDIR11基因全长编码区的克隆
(1)取0.3克新鲜甘蔗芽，按照全式金生物科技公司的Transgen TransZol Plant试剂盒使用说明提取总RNA，再用诺唯赞生物科技公司的II 1st Strand cDNA合成试剂盒将RNA反转录为cDNA，放入-80℃备用。
(2)在甘蔗基因组数据库下载ScDIR11基因的序列，用SnapGene软件设计引物两端分别含有Bam HI和SacI的引物克隆ScDIR11基因。引物序列为：
正向引物5’-CGCGGATCCATGGCCAAAAGCAAGCTTAGTACC-3’
反向引物5’-CGCGAGCTCCTACACGCGCAGGTGCA-3’
(3)根据已得到的cDNA模板PCR扩增ScDIR11基因的cDNA片段。
(4)反应体系为：加入100ng的cDNA为模板，加入10μL的Prime STAR Max Premix，正反向引物各1μL，最后ddH2O补足到20μL；PCR反应程序：95℃预变性5min，95℃变性15sec，60℃退火20sec，72℃延伸30sec，进行30个循环，72℃再延伸2min。
(5)PCR产物用琼脂糖凝胶电泳检测，如图1所示，获得552bp的ScDIR11基因片段。
实施例2遗传转化质粒pCAMBIA3300-Ubi-ScDIR11-NOS的构建
构建过程如图2所示，具体按以下操作进行：
(1)将Ubi片段和pCAMBIA3300-NOS载体用限制性内切酶Bam HI和Hind III连接得到质粒pCAMBIA3300-Ubi-NOS，酶切验证结果如图3所示。
(2)将PCR扩增的ScDIR11基因与pCAMBIA3300-Ubi-NOS质粒连接，得到遗传转化载体pCAMBIA3300-Ubi-ScDIR11-NOS，酶切验证结果如图4所示。
(3)重组载体送于上海生工生物公司进行测序，-80℃保存测序正确的pCAMBIA3300-Ubi-ScDIR11-NOS载体备用。
实施例3 ScDIR11基因遗传转化甘蔗
(1)用农杆菌介导法分别将pCAMBIA3300-Ubi-NOS空质粒和pCAMBIA3300-Ubi-ScDIR11-NOS遗传转化质粒导入甘蔗胚性愈伤组织(感黑穗病的甘蔗品种ROC 22)，Bar基因为pCAMBIA3300质粒上的筛选标记基因。
(2)转基因甘蔗的鉴定
通过PCR和qRT-PCR检测表明一个转基因甘蔗株系的DNA中含有Bar基因(图5)；另一个株系的DNA中ScDIR11基因的表达量显著提升，表明ScDIR11基因成功在甘蔗中过表达(图6和图7)。
实施例4检测甘蔗的木质素含量
分别取转基因甘蔗和非转基因甘蔗的芽，80℃干燥两天，用北京索莱宝科技公司的木质素检测试剂盒测定木质素含量。如图8所示，转基因甘蔗的木质素含量显著高于未转基因甘蔗和转入空载甘蔗的芽，再次表明ScDIR11基因成功过表达于转基因甘蔗株系。
实施例5检测甘蔗对甘蔗鞭黑粉菌的抗性
(1)将甘蔗鞭黑粉菌单倍体JG35、JG36接种于液体YEPS培养基中，28℃，200rpm摇床培养至OD600时的吸光值为1.0。收集菌体，重悬于与菌液等体积的无菌水中，形成侵染液。
(2)清洗好生根的甘蔗组织培养幼苗，放入侵染液，浸泡根部，28℃光照培养室放置培养3天。浸泡完后种植于育苗基质上，在光/暗为16h/8h，温度28℃，湿度为80％的人工气候室中培养统计甘蔗黑穗病发病情况。
(3)如图9所示，接种后第41天，非转基因甘蔗最先出现发病植株；侵染后的第46天，在35S::00甘蔗株系中出现发病植株；第61天，35S::ScDIR11两个甘蔗株系中出现发病植株。甘蔗鞭黑粉菌接种150天后，非转基因甘蔗植株和35S::00株系的发病率分别为37/50(74.0％)和36/50(72.0％)，35S::ScDIR11甘蔗株系的发病率为4/35(11.4％)，无菌水浸泡的对照组甘蔗植株在统计时间内均未发病。
实施例6检测甘蔗鞭黑粉菌接种后甘蔗茎尖生长点的黑粉菌含量
(1)在甘蔗鞭黑粉菌接种甘蔗幼苗150天后，取甘蔗的茎尖生长点，提取甘蔗总DNA，以DNA为模板，用特异性引物bE4和bE8检测甘蔗鞭黑粉菌。引物序列为：
引物bE4 5’-CGCTCTGGTTCATCAACG-3’
引物bE8 5’-TGCTGTCGATGGAAGGTGT-3’
(2)如图10所示，未发病的过表达ScDIR11基因的甘蔗也有甘蔗鞭黑粉菌特异基因片段的表达，但其表达量显著低于未过表达ScDIR11基因的甘蔗，表明ScDIR11基因的过表达抑制了甘蔗鞭黑粉菌的增殖。
Claims (7)
1.甘蔗ScDIR11基因，其特征在于具有序列表SEQ.ID.NO.1的碱基序列。
2.含有权利要求1所述甘蔗ScDIR11基因的遗传转化质粒。
3.权利要求2所述遗传转化质粒的构建方法，其特征在于：以单子叶植物的强启动子Ubi作为遗传转化载体的启动子，将Ubi片段和pCAMBIA3300-NOS载体用限制性内切酶BamHI和Hind III连接得到质粒pCAMBIA3300-Ubi-NOS，将PCR扩增的甘蔗ScDIR11基因与pCAMBIA3300-Ubi-NOS质粒连接，即得到pCAMBIA3300-Ubi-ScDIR11-NOS。
4.甘蔗ScDIR11基因在调控甘蔗木质素产生中的应用。
5.甘蔗ScDIR11基因在防治甘蔗黑穗病中的应用。
6.甘蔗ScDIR11基因在抗黑穗病甘蔗育种中的应用。
7.一种获得高抗黑穗病甘蔗的方法，其特征在于：将权利要求2所述的遗传转化质粒通过根癌农杆菌介导法遗传转化至甘蔗中。
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