CN106834306A - 烟草C2H2型锌指蛋白基因Nt540的应用 - Google Patents

烟草C2H2型锌指蛋白基因Nt540的应用 Download PDF

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CN106834306A
CN106834306A CN201710157893.4A CN201710157893A CN106834306A CN 106834306 A CN106834306 A CN 106834306A CN 201710157893 A CN201710157893 A CN 201710157893A CN 106834306 A CN106834306 A CN 106834306A
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徐莺
时小东
任学良
赵杰宏
王灵慧
田银帅
邹颉
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Guizhou Institute of Tobacco Science
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Abstract

本发明公开了烟草C2H2型锌指蛋白基因Nt540的应用。本发明提供了一种基因片段，其核苷酸序列如SEQ ID NO.1所示。本发明还公开了包含前述基因片段的重组载体、重组菌以及它们的用途。本发明通过转入Nt540基因以及重组菌，有效提升了烟草的品质，为烟草品种改良提供了新材料和新途径，具有较强的应用前景。

Description

烟草C2H2型锌指蛋白基因Nt540的应用
技术领域
本发明涉及基因工程领域，具体涉及烟草C2H2型锌指蛋白基因Nt540的应用。
背景技术
烟草是一种以吸食为主要目的特殊经济作物,香气足是优质烟叶必备具备品质之一，具体表现为充足纯正、劲头适中、吃味醇和、余味舒适、吸食安全性高等。影响烟叶香气因素很多，研究显示，烟叶表皮毛能分泌精油、树脂、蜡质等成分，这些物质对烟草的抗虫性有重要作用，同时与烟草的香气和吃味密切相关。据报道，存于烟草叶片和烟气中的化学成分共计5289种。在众多组分中,影响烟草的品质主要是那些形成香味和吃味的物质,如含氮化合物、碳水化合物、烟草生物碱等。一般来说,一定范围内糖含量高的烟叶品质好。氨基酸则与烟叶的香味、吃味关系密切，是形成烟叶香气物质的前体。所以，烟叶中的化学成分种类和数量直接影响到烟叶的内在质量、决定烟叶的香气、品质。
相对于在抗病毒、抗旱、抗虫转基因烟草品种培育的方面较的投入和成果，基因工程技术在改善烟草品质方面的应用研究较少，目前主要集中在针对某一类具体物质如萜类、胡萝卜素类或钾离子等物质的含量进行调控，而协调烟草碳氮比、降低烟碱等方面研究较少，特别是在整体水平上调控烟叶组成，从而提高相关品质的研究较少。
发明内容
本发明的目的是提供了一种新的基因，并通过将其转基因的方式来提高植物组织培养分化效率。
本发明首先提供了一种基因片段，其核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种重组载体，它包括SEQ ID NO.1所示的核苷酸序列。优选地，所述重组载体是重组pBI121载体。
本发明还提供了一种重组菌，它包含前述重组载体.优选地，所述重组菌为重组农杆菌，进一步优选为重组农杆菌EHA105。
本发明还提供了一种蛋白，其氨基酸序列如SEQ ID NO.2所示.
本发明还提供了前述的基因片段、重组载体、重组菌、蛋白在提高植物的叶片表皮毛数量、游离氨基酸含量、还原糖含量和/或总糖含量中的用途。
其中，所述植物为烟草。
本发明还提供了一种提高植物的叶片表皮毛数量、游离氨基酸含量、还原糖含量和/或总糖含量的方法，取前述的基因片段、重组载体、重组菌，转入植物中，即可。
其中，所述植物为烟草。
本发明发现将Nt540基因转入烟草植株过表达后，可显著改变烟叶品质相关成分的含量。除烟碱外，过表达该基因的烟草植物的叶片表皮毛数量、游离氨基酸含量、总糖和还原糖的含量都显著提高，说明该基因的导入能够增加叶片表皮毛数量、改变烟叶的化学组成，从而进一步改变烟叶的品质。单基因导入使得多种成分发生变化，在烟叶中并不多见。
本发明制备得到了Nt540基因以及重组菌，将其转入植物中，可以有效提升烟草的品质，本发明为烟草品种改良提供了新材料和新途径，具有较强的应用前景，经济效益良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1.Nt540基因PCR产物电泳检测结果，其中M为分子量标准；1为空白对照，2和3为Nt540片段；
图2.为烟草抗性植株再生情况。其中，A为抗性芽分化情况；B为抗性植株生长；C为抗性植株生根情况；
图3.稳定表达植株中Nt540基因表达的实时定量PCR分析.其中A为野生型，B为超表达Nt540型。
具体实施方式
生物材料：烟草(Nicotianatabacco)、大肠杆菌(Escherichia coli)菌株DH5α、根癌农杆菌(Agrobacterium tumefaciens)EHA105、载体pBI121、载体pCAMBIA1301由生物资源与生态环境教育部重点实验室(四川大学)保存。
所用试剂均为市售的进口或国产分析纯试剂。
实施例1本发明烟草Nt540基因的制备及其应用
(1)基因片段和载体的构建
提取烟草总RNA，并取少量RNA用于琼脂糖凝胶电泳，叶片总RNA电泳结果表明，总RNA的28S rRNA与18S rRNA之间的亮度比约为2:1，无明显拖尾，也看不见明显的DNA和蛋白质污染，说明RNA提取的结果较好，降解少，分子完整，表明所提mRNA纯度高，可用于反转录，获得cDNA进行后续试验。
利用含有XbaI和XmaI酶切位点的特异性引物(F:gctctagaat ggagaaaacagacagagaaa ct；R：tcacaagtgt agatctaaac tcacatg)，以烟草cDNA为模板进行PCR扩增目的基因的开放阅读框(ORF)。其PCR产物用1％琼脂糖凝胶电泳分析，分子量约为700bp左右，与预计片段大小相符合(附图1)。
将PCR产物胶回收后连接T载体转入DH5α感受态，获得阳性克隆进行测序。测序结果显示目的基因Nt540的核苷酸序列为：
前述核苷酸序列所述的NtZFP8基因可以通过采用上述方法获得，也可以直接合成。
将测序正确的大肠杆菌质粒和pBI121质粒经过限制性内切酶XbaI和XmaI双酶切，回收目的条带。用T4连接酶连接目的条带，16℃连接过夜获得重组子后，将重组子转入大肠杆菌感受态细胞获得转化子。以转化子质粒为模板，用目的基因特异性引物对其进行扩增获得一条大小一致的目的片段，初步表明转化子中含有目的序列。随后用限制酶XbaI和XmaI进行双酶切，电泳检测获得一条大片段和目的条带大小的小片段。证明pBI121载体已与目的基因Nt540相连，表达载体构建成功。
提取转化子的质粒，将其转化农杆菌EHA105感受态后，为鉴定转化是否成功，挑取单菌落进行菌落PCR。菌落PCR结果显示出现了与目的基因Nt540的大小一致的目的带，对条带进行测序，与Nt540序列相一致，表明构建正确。
(2)转基因植物的制备及其性质检测
先将烟草叶盘预培养2-3天，然后用含有植物表达载体的根癌农杆菌分别进行侵染。共培养3-4天后，将叶盘取出，用无菌水洗菌，然后再转到筛选培养基进行筛选及分化培养，约3周左右开始分化出抗性芽。待抗性芽长至3-4cm高时，将其从外植体基部切下，接种到生根培养基上进行生根培养。待小植株根系发达后，进行驯化移栽(图2)。得到大量移栽成活的抗性植株。将PCR检测呈阳性的植株移栽到大花盆中，直到收获种子，多次种植，直到获得Nt540稳定遗传的转基因植株。同时，对获得的稳定转基因植株进行荧光定量PCR验证，结果表明其Nt540基因表达量明显高于野生型对照，说明基因过表达成功(附图3)，证明本发明构建得到了含有Nt540基因的转基因烟草。
目的基因Nt540表达的蛋白质的氨基酸序列为：
MEKTDRETRDFMNVESFSQLPFIRPAPAKEKAIRLFGKEFGTAGDSMAATDESESIETNPSQEEIKDHISNNSESSRKFECHYCCRNFPTSQALGGHQNAHKRERQHAKRAHLQSAMVHGALTEANIYGIMNYHRLGSAPTAAYHHHYTTNNINNATRFYGSQHHVNSTVTTPYNSHQTPINGSPLALWRIPAATNVHHTSSSSPNFGRERSMNMQPLPVFAANNEDFKPSPIINSSSSQRGFGYKTKAGVKDHVSLDLHL
进一步观察含有Nt540基因的转基因烟草的叶片表皮毛，发现与非转基因野生型烟草植株相比，过表达的转基因烟草叶片上的表皮毛数量明显更多。
对烟草叶片表皮毛数量和成分进行测定，发现转基因植株的表皮毛数量、游离氨基酸、还原糖和总糖含量显著高于野生型对照，而烟碱含量无明显变化(表1)。
表1烟草叶片成分测定结果
实验结果说明，本发明Nt540基因可以有效提高烟草的表皮毛数量，能够更有效对的抵御害虫，还能提高香味，还可以显著提高其游离氨基酸、还原糖和总糖的含量，提升烟草的品质，而有害物质烟碱的含量没有明显变化。
综上，本发明Nt540基因可以有效提高烟草的品质，制备的转基因烟草的表皮毛数量多，游离氨基酸、还原糖和总糖的含量高，应用前景良好。
SEQUENCE LISTING
<110> 贵州省烟草科学研究院
四川大学
<120> 烟草C2H2型锌指蛋白基因Nt540的应用
<130> GY007-17P1103
<160> 4
<170> PatentIn version 3.5
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<213> 烟草C2H2型锌指蛋白基因Nt540的应用
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<213> 目的基因Nt540表达的蛋白质的氨基酸序列
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Gly Ser Gln His His Val Asn Ser Thr Val Thr Thr Pro Tyr Asn Ser
165 170 175
His Gln Thr Pro Ile Asn Gly Ser Pro Leu Ala Leu Trp Arg Ile Pro
180 185 190
Ala Ala Thr Asn Val His His Thr Ser Ser Ser Ser Pro Asn Phe Gly
195 200 205
Arg Glu Arg Ser Met Asn Met Gln Pro Leu Pro Val Phe Ala Ala Asn
210 215 220
Asn Glu Asp Phe Lys Pro Ser Pro Ile Ile Asn Ser Ser Ser Ser Gln
225 230 235 240
Arg Gly Phe Gly Tyr Lys Thr Lys Ala Gly Val Lys Asp His Val Ser
245 250 255
Leu Asp Leu His Leu
260
<210> 3
<211> 32
<212> DNA
<213> 目的基因的扩增引物F
<400> 3
gctctagaat ggagaaaaca gacagagaaa ct 32
<210> 4
<211> 27
<212> DNA
<213> 目的基因的扩增引物R
<400> 4
tcacaagtgt agatctaaac tcacatg 27

Claims (10)

1.一种基因片段，其特征在于：其核苷酸序列如SEQ ID NO.1所示。
2.一种重组载体，其特征在于：它包括SEQ ID NO.1所示的核苷酸序列。
3.根据权利要求2所述的重组载体，其特征在于：所述重组载体是重组pBI121载体。
4.一种重组菌，其特征在于：它包含权利要求3或4所述的重组载体。
5.根据权利要求2所述的重组菌，其特征在于：所述重组菌为重组农杆菌，优选为重组农杆菌EHA105。
6.一种蛋白，其特征在于：其氨基酸序列如SEQ ID NO.2所示。
7.权利要求1～6任意一项所述的基因片段、重组载体、重组菌、蛋白在提高植物的叶片表皮毛数量、游离氨基酸含量、还原糖含量和/或总糖含量中的用途。
8.根据权利要求7所述的用途，其特征在于：所述植物为烟草。
9.一种提高植物的叶片表皮毛数量、游离氨基酸含量、还原糖含量和/或总糖含量的方法，其特征在于：取权利要求1～5任意一项所述的基因片段、重组载体、重组菌，转入植物中，即可。
10.根据权利要求9所述的方法，其特征在于：所述植物为烟草。
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104059925A (zh) * 2014-06-10 2014-09-24 浙江大学 拟南芥锌指蛋白基因gis2在促进烟草腺毛生长中的应用

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* Cited by examiner, † Cited by third party
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CN104059925A (zh) * 2014-06-10 2014-09-24 浙江大学 拟南芥锌指蛋白基因gis2在促进烟草腺毛生长中的应用

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