CN116286710A - 一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8及其应用 - Google Patents
一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8及其应用 Download PDFInfo
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Abstract
本发明公开了一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8及其应用，该烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8来源于烟草，其核苷酸序列如SEQ ID NO.1所示，包括894bp个碱基。本发明通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除NtXTH8基因的CRISPR/Cas9编辑载体，经遗传转化后获得了NtXTH8基因发生编辑突变体植株。在现蕾期、成熟期进行NtXTH8基因编辑植株与对照植株农艺性状调查，NtXTH8基因编辑植株中株高、腰叶长、腰叶宽等农艺性状进行测定，均低于对照；对精氨酸含量进行测定，发现成熟期基因编辑植株中精氨酸含量显著低于对照。
Description
技术领域
本发明涉及植物基因技术领域，特别涉及一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8及其应用。
背景技术
木葡聚糖内转葡萄糖酶/水解酶(XTH)基因家族是GH16的一个亚家族，基于碳水化合物活性酶(CAZy)分类。植物木葡聚糖内转葡糖基酶/水解酶蛋白8(NtXTH8)可能参与生长组织的细胞壁构建，对植物生长发育、成熟软化有重要影响。XTH蛋白参与了植物的多个生长发育过程，如根的伸长、叶脉分化、花的生长发育、果实成熟、花瓣衰老等过程。同时XTH基因家族以不同的形式参与到植物的生物和非生物胁迫响应当中，对提高植物的抗逆能力具有重要作用。
拟南芥中XTHs基因研究表明该基因是根系发育过程中的一个重要响应基因。拟南芥AtXTH21主要在根系和花中表达，之后通过过表达和T-DNA插入突变证实了其通过改变根部纤维素沉积以及细胞壁延展性在主根发育中起重要作用。水稻OsXTH8的表达会被GA诱导上调，且采用RNAi干扰后，水稻生长受到抑制，通过实验充分证明了OsXTH8在水稻茎叶生长发育中的重要作用。
烟草作为一种以收获营养器官为主的嗜好性叶用经济作物，研究烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因对生长发育的分子机制具有重要意义。在烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因克隆与功能研究方面，从普通烟草中共鉴定出56个NtXTH基因，根据系统发育关系可将56个NtXTH基因分为I/II组和III组两个亚家族。
发明内容
本发明所要解决的技术问题是提供一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8及其应用，为研究烟草生长发育相关基因功能提供遗传材料和理论依据。
本发明所要解决的技术问题是通过以下技术方案来实现的：
一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8，NtXTH8基因来源于烟草，其核苷酸序列如SEQ ID NO.1所示，包括894bp个碱基。
优选地，NtXTH8基因的编码蛋白的氨基酸序列如SEQ ID NO.2所示，包括298个氨基酸。
一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8在烟草生长发育中的应用。
优选地，将NtXTH8基因编辑后，NtXTH8基因编辑植株的长势弱于对照植株。
优选地，NtXTH8基因编辑植株现蕾期进行株高、腰叶长、腰叶宽的测定，均低于对照植株；成熟期进行有效叶数、打顶株高、腰叶宽、自然株高及自然叶数的测定，均不同程度低于对照植株；成熟期精氨酸含量显著低于对照植株。
优选地，基因编辑是通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除NtXTH8基因的CRISPR/Cas9编辑载体，经遗传转化后获得了突变体编辑植株。
本发明上述技术方案，具有如下有益效果：
本发明通过同源克隆方法克隆一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8。
本发明通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除NtXTH8基因的CRISPR/Cas9编辑载体，经遗传转化后获得了NtXTH8基因发生编辑的红花大金元突变体植株。
本发明通过在正常条件下，现蕾期、成熟期进行NtXTH8基因编辑植株与对照(未转化)植株农艺性状调查，NtXTH8基因编辑植株中株高、腰叶长、腰叶宽等农艺性状进行测定，均低于对照(未转化)中的。同时对素材中精氨酸含量进行测定，发现成熟期基因编辑植株中精氨酸含量显著低于对照。
综上所述，利用CRISPR/Cas9介导的基因编辑技术敲除NtXTH8基因获得影响植株生长发育的基因编辑植株，这为烟草木葡聚糖内转葡糖基酶/水解酶蛋白研究及烟草生长发育研究提供遗传材料和理论依据。
附图说明
被结合在说明书中并构成说明书的一部分的附图示出了本发明的实施例，并且连同其说明一起用于解释本发明的原理。
图1为NtXTH8基因克隆胶图；
图2为对照(未转化)植株和基因编辑植株在现蕾期正常条件下，进行植株株高、腰叶长、腰叶宽的测定。
图3为对照(未转化)植株和基因编辑植株在成熟期正常条件下，进行植株有效叶数、打顶株高、腰叶长、腰叶宽、自然株高及自然叶数的测定。
图4为对照(未转化)植株和基因编辑植株在成熟期正常条件下，进行精氨酸含量的测定。
具体实施方式
现在将参照附图来详细描述本发明的各种示例性实施例。应注意到：除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本发明的范围。
实施例1
本实施例主要就烟草木葡聚糖内转葡糖基酶/水解酶蛋白相关基因NtXTH8的获得过程，简要介绍如下。
以栽培种烟草红花大金元叶片为样品，利用RNA提取试剂盒提取烟草叶片总RNA，反转录为cDNA备用：
按照植物RNA提取试剂盒说明书提取烟草总RNA。
1μg从叶片中提取总RNA用于反转录，转录体系如下：
Total RNA 1μg；
Oligo(dT)(10μM) 1.5μL；
ddH2O up to 15μL。
将上述体系混匀后置于PCR中，70℃保温5min，去除后立即置于冰上5min，之后向体系中加入以下试剂：
上述体系放入PCR仪中，42℃65min，65℃10min，4℃保温，之后置于-20℃冰箱中保存使用。
通过同源比对的方法，参考拟南芥基因的序列及已知烟草部分基因序列，设计扩增引物序列如下：
F：5’-ATGGAGAAAATGGCTTCTTC-3’，(SEQ ID No.3)；
R：5’-TTATTCCCATGGATTTAACC-3’；(SEQ ID No.4)。
以上述所制备cDNA为模板，利用上述引物进行PCR扩增：
扩增体系(50μL)：
混匀离心后进行PCR扩增，PCR反应条件为：95℃10sec，52℃30sec，72℃2.5min，共30个循环；72℃10min；12℃Hold。
对扩增产物进行提纯后测序，获得烟草木葡聚糖内转葡糖基酶/水解酶蛋白相关基因NtXTH8序列，其碱基序列如SEQ ID No.1所示，共包括894bp个碱基(结果如图1所示)。对该基因序列进行翻译后，其所编码蛋白序列如SEQ ID No.2所示，共包括298个氨基酸，进一步对比分析表明，该蛋白含有同源性很高的序列，高度保守。
实施例2
利用实施例1中所获得烟草木葡聚糖内转葡糖基酶/水解酶蛋白相关基因NtXTH8，本发明进一步构建了CRISPR/Cas9载体，以及利用叶盘法转化获得基因编辑植株。
选择NtXTH8基因中较特异的23nt核苷酸序列(SEQ ID No.5)为CRISPR/Cas9的引导序列，并将该序列片段与CRISPR/Cas9载体(由西南大学提供)进行连接，获得转化的克隆子，进行PCR扩增检测，后将PCR阳性克隆送测序公司进行测序确认，最后得到CRISPR/Cas9-NtXTH8编辑载体。
利用上一步所构建的CRISPR/Cas9-NtXTH8编辑载体质粒，以红花大金元为例，进行遗传转化试验，以敲除植物体内的烟草木葡聚糖内转葡糖基酶/水解酶蛋白相关基因NtXTH8。
将获得的经农杆菌介导转化NtXTH8基因的再生植株，后进行转化植株叶片取样，送华大基因进行分子检测，确定获得NtXTH8基因编辑植株。
实施例3
种植编辑植株与对照植株，现蕾期与成熟期进行烟草植株株高、腰叶长、腰叶宽进行测定，编辑素材中株高、腰叶长、腰叶宽均低于对照素材(结果如图2和图3所示)。同时测定成熟期叶片中精氨酸含量，编辑素材中显著低于对照(结果如图4所示)。
虽然本发明已以实施例公开如上，然其并非用于限定本发明，任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种不同的选择和修改，因此本发明的保护范围由权利要求书及其等同形式所限定。
Claims (6)
1.一种烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8，其特征在于，NtXTH8基因来源于烟草，其核苷酸序列如SEQ ID NO.1所示，包括894bp个碱基。
2.根据权利要求1所述的烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8，其特征在于，NtXTH8基因的编码蛋白的氨基酸序列如SEQ IDNO.2所示，包括298个氨基酸。
3.根据权利要求1-2任一所述的烟草木葡聚糖内转葡糖基酶/水解酶蛋白基因NtXTH8在烟草生长发育中的应用。
4.根据权利要求3所述的应用，其特征在于，将NtXTH8基因编辑后，NtXTH8基因编辑植株的长势弱于对照植株。
5.根据权利要求3所述的应用，其特征在于，NtXTH8基因编辑植株现蕾期进行株高、腰叶长、腰叶宽的测定，均低于对照植株；成熟期进行有效叶数、打顶株高、腰叶宽、自然株高及自然叶数的测定，均不同程度低于对照植株；成熟期精氨酸含量显著低于对照植株。
6.根据权利要求3所述的应用，其特征在于，基因编辑是通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除NtXTH8基因的CRISPR/Cas9编辑载体，经遗传转化后获得了突变体编辑植株。
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