CN113897380A - 参与烟草盐胁迫反应的基因及其应用 - Google Patents

参与烟草盐胁迫反应的基因及其应用 Download PDF

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CN113897380A
CN113897380A CN202111459338.XA CN202111459338A CN113897380A CN 113897380 A CN113897380 A CN 113897380A CN 202111459338 A CN202111459338 A CN 202111459338A CN 113897380 A CN113897380 A CN 113897380A
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许力
许�永
王凯
高茜
杨文武
米其利
李雪梅
向海英
曾婉俐
蒋佳芮
鲁逸飞
邓乐乐
张建铎
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China Tobacco Yunnan Industrial Co Ltd
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Abstract

本发明公开了一种参与烟草盐胁迫反应的基因，所述基因的核苷酸序列如SEQ ID NO.1所示。本发明将得到的基因构建到基因编辑的表达载体，将获得的基因编辑的烟草植株进行盐胁迫反应后其过氧化物酶、脯氨酸含量提高，特别是脯氨酸含量提高明显。

Description

参与烟草盐胁迫反应的基因及其应用
技术领域
本发明属于植物基因工程技术领域，具体涉及参与烟草盐胁迫反应的基因及其应用。
背景技术
CIPK(CBL-interacting protein kinase)是一类具有生物活性的丝/苏氨酸蛋白激酶，通过与类钙调神经素B亚基蛋白(Calcineurin B-like protein，CBL)相互作用从而调节植物在适应或抵制非生物逆境胁迫中的生理活动。目前已经从许多植物中克隆到这个家族基因(Tang等2014；Weinl等2009)。CIPK在植物响应高盐、低温、高温和低钾信号转导过程中发挥重要作用。野大麦HbCIPK2在干旱、高盐和ABA胁迫下表达增强(Li等2012)。拟南芥和马铃薯中CIPK23参与低钾胁迫的反应(Xu等2006；王婉晶，2017)，烟草NtCIPK23受到干旱、盐胁迫诱导(闫亚飞2012)。目前在一些植物(水稻、拟南芥、马铃薯等)中CIPK23基因已经被报道相关功能，但烟草CIPK23基因在盐胁迫中的功能未知。
为解决上述问题提出本发明。
发明内容
本发明的目的是获得烟草CIPK23基因编辑材料并进行功能分析。本发明从烟草中克隆得到CIPK基因，见所述基因的核苷酸序列如SEQ ID NO.1所示，核苷酸序列全长1314bp碱基。具体技术方案是，参考GenBank收录烟草CIPK序列，登陆号是XM_016583370.1，采用Primer5.0软件设计引物，见表1。扩增反应程序为：95℃预变性5min；95℃变性30s；55℃退火30s；72℃延伸90s；35个循环。目的片段纯化后与pMD19-T载体16℃连接过夜，连接产物转化大肠杆菌DH5α感受态，随后在涂有氨苄青霉素的LB平板上进行筛选，采用菌落PCR检测阳性克隆。检测后随机选取三个独立的阳性克隆送生物技术公司进行测序。本发明烟草CIPK基因的核苷酸序列如SEQ ID NO.1所示，该烟草CIPK23基因核苷酸序列全长1314bp。随后将该基因构建到基因编辑的表达载体，利用农杆菌介导的方法进行烟草红花大金元遗传转化，利用获得的基因编辑材料进行功能研究特别是盐胁迫研究。
本发明第一方面公开了一种参与烟草盐胁迫反应的基因，核苷酸序列如SEQ IDNO.1所示，包括1314bp个碱基。
优选地，所述参与烟草盐胁迫反应的基因的编码蛋白。
优选地，所述编码蛋白的氨基酸序列如SEQ ID NO.2所示，包括437个氨基酸。
本发明第二方面公开了所述的参与烟草盐胁迫反应的基因的应用，将所述基因构建到基因编辑的表达载体，利用农杆菌介导的方法进行烟草遗传转化，然后将获得的基因编辑的烟草植株进行烟草盐胁迫反应。
优选地，所有烟草植株、包括对照烟草植株(即普通烟草植株)与基因编辑后的烟草植株盐胁迫后其过氧化物酶含量、脯氨酸含量提高，特别是脯氨酸含量提高明显；但叶绿素含量低于正常生长的烟草植株和基因编辑后的烟草植株。
优选地，所述基因编辑后的烟草植株盐胁迫后，其过氧化物酶含量和脯氨酸含量均高于对照烟草植株，特别是脯氨酸含量明显较高；但过氧化氢浓度略低于对照烟草植株。
本发明的有益效果：
1、本发明通过克隆得到烟草CIPK23基因，其核苷酸序列如如SEQ ID NO.1所示，核苷酸序列全长1314bp个碱基。
2、本发明将得到的CIPK23基因构建到基因编辑的表达载体，利用农杆菌介导的方法进行烟草遗传转化，然后将获得的基因编辑的烟草植株进行烟草盐胁迫反应后，其过氧化物酶、脯氨酸含量提高，特别是脯氨酸含量提高明显。
附图说明
图1为本发明的CIPK23基因的电泳图。
图2为CIPK23基因编辑的烟草植株盐处理表型检测；HD为对照样即普通烟草植株，T1-T3为基因编辑后的烟草植株；其中左边为正常生长，右边为盐处理后。
图3为CIPK23基因编辑的烟草植株盐处理后叶绿素含量测定；HD为对照样即普通烟草植株，T1-T3为基因编辑后的烟草植株；其中左边为正常生长，右边为盐处理。
图4为CIPK23基因编辑的烟草植株盐处理后过氧化物酶(POD)含量测定；HD为对照样即普通烟草植株，T1-T3为基因编辑后的烟草植株；其中左边为正常生长，右边为盐处理。
图5为CIPK23基因编辑的烟草植株盐处理后过脯氨酸含量测定；HD为对照样即普通烟草植株，T1-T3为基因编辑后的烟草植株；其中左边为正常生长，右边为盐处理。
图6为CIPK23基因编辑的烟草植株盐处理后过氧化氢浓度测定；HD为对照样即普通烟草植株，T1-T3为基因编辑后的烟草植株；其中左边为正常生长，右边为盐处理。
具体实施方式
以下通过实施例来详细说明本发明的技术方案，以下的实施例仅是示例性的，仅能用来解释和说明本发明的技术方案，而不能解释为是对本发明技术方案的限制。在本申请的各实施例中，没有注明具体技术或条件者，按照本领域内现有技术或条件进行，所使用的材料或设备未注明生产厂商者，均为可以通过购买获得的常规产品。本发明除非另有说明，否则百分号为体积百分数，比例为体积比。
实施例1：基因克隆及烟草植株基因编辑
1、基因克隆
取0.5g烟草新鲜叶片，采用Trizol法提取烟草细胞的总RNA，然后采用TaKaRa公司的cDNA合成试剂盒合成cDNA，进一步采用Primer5.0软件设计引物对cDNA进行PCR扩增，引物见下表1。
扩增反应程序为：95℃预变性5min；95℃变性30s；55℃退火30s；72℃延伸90s；35个循环。目的片段纯化后与pMD19-T载体16℃连接过夜，连接产物转化大肠杆菌DH5α感受态，随后在涂有氨苄青霉素的LB平板上进行筛选，采用菌落PCR检测阳性克隆。检测后随机选取三个独立的阳性克隆送生物技术公司进行测序，得到的烟草CIPK23基因的核苷酸序列如SEQ ID NO.1所示，该烟草CIPK23基因核苷酸序列全长1314bp碱基。
表1引物列表
Figure BDA0003389267060000041
图1为得到的CIPK23基因的电泳图。
2、目的基因连接表达载体
将上述转化好的T-载体与基因编辑的表达载体进行双酶切，回收目的基因和表达载体，然后用连接酶连接，将连接后的重组表达载体转入大肠杆菌DH5α的感受态细胞，对转化后的大肠杆菌单菌落进行PCR扩增，然后挑取阳性菌落扩繁、提取质粒进行酶切和测序来检测是否构建成功。
3、冻融法转化农杆菌和PCR检测
取200μL感受态细胞，室温融化后加入10μL构建好的重组质粒DNA，混匀后冰浴30min，液氮中速冻1min，37℃水浴5min，然后加入500μL含利福平和链霉素的LB培养基，28℃慢速振荡培养4h，10000r/min离心1min集菌，弃上清，加入500μL含利福平和链霉素的LB培养基重新悬浮细胞，涂布于含有50μg/mL利福平、50μg/mL链霉素和50μg/mL卡那霉素的平板上，28℃避光培养约48h。待平板上长出的单菌落，直接用灭菌的牙签蘸取单菌落，在PCR体系中晃动几下后进行PCR扩增反应。
Figure BDA0003389267060000042
Figure BDA0003389267060000051
1.0％的琼脂糖凝胶电泳检测PCR产物结果：若空白对照没有条带，但转录PCR产物有明亮且大小正确的条带，证明转化成功。
4、农杆菌转化烟草方法
4.1烟草无菌苗的培养和预培养
选取籽粒饱满无病虫害的烟草种子装入1.5mL EP管中先用70％的乙醇消毒30s，无菌水冲洗3-5次，然后用1mL30％次氯酸钠溶液消毒5min，吸出消毒液，再加入1mL30％次氯酸钠溶液消毒25min，消毒期间都要不断地振荡EP管。最后用无菌水反复清洗6-7次后，吸干种子表面水分，布于MS培养基上，于光照培养箱最大光强度、温度20℃、光照16h/d培养30d左右备用。选取约30d苗龄且长势良好的烟草无菌苗，用灭菌刀片将烟草叶片切下，放置在预培养基上培养2d(注意培养基要倒置，即叶的正面朝下)。
4.2侵染菌液的制备
将含有目的基因的农杆菌在固体LB培养基上划板，28℃下暗培养2d。用灭菌牙签挑取菌落，接种于2mL液体LB培养基中，28℃下振荡培养过夜(12h左右)。活化过夜的农杆菌，按1:50的比例，取100μL稀释到5mL LB培养基中，继续培养至OD600值为0.5(约3h检测一次)。取培养物1mL置于无菌离心管中，12000r/min离心1min，弃上清。加入100mL的MS0培养基，混匀备用。
4.3侵染叶片、共培养和分化培养
将在预培养基上培养2d的烟草叶片切成1cm2左右的叶盘，置于悬菌液中(MS0悬浮，可以稀释50-100倍)浸泡3-5min。然后取出，用无菌滤纸吸去其表面的液体。将侵染过的叶盘分别接种在覆有一层滤纸的共培养基上面，放到恒温培养箱20℃暗培养2d。用100mL含有200μL头孢霉素，100μL羧苄青霉素的无菌水清洗4min，重复一次后再用无菌水清洗8min，最后用无菌滤纸吸去其表面的液体再转入分化培养基进行分化培养，前期3d继代一次，每次继代需在无菌条件下进行，连续继代3次后就每隔两周继代一次。
4.4生根培养和繁殖
待抗性芽长至1-2cm时，在超净台上切去芽基部的所有愈伤组织及基部叶片，植于生根培养基上。待根长至2-3cm时，取出无菌苗，轻轻打碎固体培养基，洗去残留的培养基，去掉下部叶，然后将无菌苗植入土壤，室内培养大约一周后移到室外(最初的3d应在阴暗处生长且要盖上透明塑料)。经过PCR检测为阳性的烟草植株进行繁殖到T3代。
实施例2：基因编辑后的烟草植株盐胁迫实验
1、基因编辑后的烟草植株盐处理后表型观察
采用漂浮育苗的方法，对3个基因编辑后的烟草植株和对照样(普通烟草植株)进行培养，30天后种植在培养盘中进行盐胁迫实验；使用500mM氯化钠溶液处理，处理15天后进行照相和生理指标的测定，结果如图2所示。从图2可以明显看到盐处理15天后三个基因编辑后的烟草植株长势明显优于对照样。
2、基因编辑后的烟草植株盐处理后的生理指标测定
选择三个基因编辑后的烟草植株进行盐处理后的生理指标进行测定，并与对照样(普通烟草植株)进行盐处理后比较，结果如图3-图6所示。由图3-图6可以看出，叶绿素含量、过氧化物酶和脯氨酸含量在盐处理15天后基因编辑后的烟草植株中明显高于对照样；而在正常的生长条件下，基因编辑后的烟草植株与对照样没有明显差异，见图3-图5。而过氧化氢含量测定结果表明在盐处理15天后基因编辑后的烟草植株中过氧化氢含量明显低于对照样；而在正常的生长条件下基因编辑后的烟草植株与对照样没有明显差异，如图6所示。
以上结果表明，烟草CIPK基因编辑后的烟草植株具有较高抵抗盐胁迫的能力。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Figure BDA0003389267060000081
Figure BDA0003389267060000091
Figure BDA0003389267060000101
Figure BDA0003389267060000111
Figure BDA0003389267060000121
序列表
<110> 云南中烟工业有限责任公司
<120> 参与烟草盐胁迫反应的基因及其应用
<130> RIB210607
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1314
<212> DNA
<213> 人工序列(CIPK23)
<400> 1
atggccgccg ctacaccacc atcttctgct tctaaaatca gaagaacaag cagcgctgat 60
ggtggtggaa gcggttcaat aattctcgga aaatatcaat taagtcacct tcttggccgt 120
ggcagcttcg caaaagttta tcatgcacga tgtttaaacg atggcacaga cgttgccatc 180
aaagttatga acaaaaacac aaccgtgaat gcctctctgg aacgagtaat catcggtgaa 240
gtctccgcca tgcatcgact taaccatcat cccaacatta tcaaactcca tgaagtcatg 300
gccacaaaga ccaaaatcta ccttgtcatg gaactcgcac ctggtggcga tcttttctcc 360
aagctcagcc gccgcggaaa attctcttac tccacagcaa gattttactt ccaccagctt 420
gtctcagctc tacacttttg ccatcaaaac ggcgtggctc accgtgatat taaaccacaa 480
aacattctcc tggacaaaga aggccagctt aaggtctctg attttggatt agccgctctt 540
tcggaacagt tcagtaacag cctcctccaa acggcttgtg gtactccagc ttatgcggct 600
ccagagattg tatacagaaa aggatatgat ggtgctaagg cggatgcatg gtcttgtggg 660
gtaatattct tttcattcct tgcaggaaaa cttccattcg atgacagcag cttaaccaaa 720
ttgcatctgg caaaacatcg tcgtgaattt caattccctg attgtgttca aaagcctgca 780
cggagtataa taaaccggct gctcgatcct aatccaacaa caaggttaag cattgaggaa 840
ttaatggagc ttccatggtt taagaaatct gcaaagaaag gcgaagagtc aaatcagaaa 900
caatttagtc aaggtatatt tgaaattaag gattgtaaaa aagaggggag aatgaatgcg 960
tttgatataa tatcaatgtc agctgggttg gatttatcag tgttatttga agcaaggttg 1020
aataagaaag agatgaggtt tacaacgact gctcaagtag ggagtattga agagaaggtg 1080
atgaaaattg ggaaaaaaga aggatataga gttgagagaa caaagggtgg gggaattggt 1140
ttggtaaaag ggagagtggc attattggtg gaaatatggg aagtgacagt agagctgtgg 1200
ttggtggaga ttaaagttgt caatggagga gtagaatttg atgaatctca ttgggaggta 1260
ctgaaagaag cgctgaaaga tattgttgtt tcatggtatg atgatgggtc ttaa 1314
<210> 2
<211> 437
<212> PRT
<213> 人工序列(CIPK23)
<400> 2
Met Ala Ala Ala Thr Pro Pro Ser Ser Ala Ser Lys Ile Arg Arg Thr
1 5 10 15
Ser Ser Ala Asp Gly Gly Gly Ser Gly Ser Ile Ile Leu Gly Lys Tyr
20 25 30
Gln Leu Ser His Leu Leu Gly Arg Gly Ser Phe Ala Lys Val Tyr His
35 40 45
Ala Arg Cys Leu Asn Asp Gly Thr Asp Val Ala Ile Lys Val Met Asn
50 55 60
Lys Asn Thr Thr Val Asn Ala Ser Leu Glu Arg Val Ile Ile Gly Glu
65 70 75 80
Val Ser Ala Met His Arg Leu Asn His His Pro Asn Ile Ile Lys Leu
85 90 95
His Glu Val Met Ala Thr Lys Thr Lys Ile Tyr Leu Val Met Glu Leu
100 105 110
Ala Pro Gly Gly Asp Leu Phe Ser Lys Leu Ser Arg Arg Gly Lys Phe
115 120 125
Ser Tyr Ser Thr Ala Arg Phe Tyr Phe His Gln Leu Val Ser Ala Leu
130 135 140
His Phe Cys His Gln Asn Gly Val Ala His Arg Asp Ile Lys Pro Gln
145 150 155 160
Asn Ile Leu Leu Asp Lys Glu Gly Gln Leu Lys Val Ser Asp Phe Gly
165 170 175
Leu Ala Ala Leu Ser Glu Gln Phe Ser Asn Ser Leu Leu Gln Thr Ala
180 185 190
Cys Gly Thr Pro Ala Tyr Ala Ala Pro Glu Ile Val Tyr Arg Lys Gly
195 200 205
Tyr Asp Gly Ala Lys Ala Asp Ala Trp Ser Cys Gly Val Ile Phe Phe
210 215 220
Ser Phe Leu Ala Gly Lys Leu Pro Phe Asp Asp Ser Ser Leu Thr Lys
225 230 235 240
Leu His Leu Ala Lys His Arg Arg Glu Phe Gln Phe Pro Asp Cys Val
245 250 255
Gln Lys Pro Ala Arg Ser Ile Ile Asn Arg Leu Leu Asp Pro Asn Pro
260 265 270
Thr Thr Arg Leu Ser Ile Glu Glu Leu Met Glu Leu Pro Trp Phe Lys
275 280 285
Lys Ser Ala Lys Lys Gly Glu Glu Ser Asn Gln Lys Gln Phe Ser Gln
290 295 300
Gly Ile Phe Glu Ile Lys Asp Cys Lys Lys Glu Gly Arg Met Asn Ala
305 310 315 320
Phe Asp Ile Ile Ser Met Ser Ala Gly Leu Asp Leu Ser Val Leu Phe
325 330 335
Glu Ala Arg Leu Asn Lys Lys Glu Met Arg Phe Thr Thr Thr Ala Gln
340 345 350
Val Gly Ser Ile Glu Glu Lys Val Met Lys Ile Gly Lys Lys Glu Gly
355 360 365
Tyr Arg Val Glu Arg Thr Lys Gly Gly Gly Ile Gly Leu Val Lys Gly
370 375 380
Arg Val Ala Leu Leu Val Glu Ile Trp Glu Val Thr Val Glu Leu Trp
385 390 395 400
Leu Val Glu Ile Lys Val Val Asn Gly Gly Val Glu Phe Asp Glu Ser
405 410 415
His Trp Glu Val Leu Lys Glu Ala Leu Lys Asp Ile Val Val Ser Trp
420 425 430
Tyr Asp Asp Gly Ser
435

Claims (6)

1.一种参与烟草盐胁迫反应的基因，其特征在于，所述基因的核苷酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的参与烟草盐胁迫反应的基因，其特征在于，所述基因的编码蛋白。
3.根据权利要求2所述的参与烟草盐胁迫反应的基因，其特征在于，所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
4.根据权利要求1-3任一所述的参与烟草盐胁迫反应的基因的应用，其特征在于，将所述基因构建到基因编辑的表达载体，利用农杆菌介导的方法进行烟草遗传转化，然后将获得的基因编辑的烟草植株进行盐胁迫反应。
5.根据权利要求4所述的应用，其特征在于，对照烟草植株与基因编辑后的烟草植株盐胁迫后其过氧化物酶含量、脯氨酸含量提高。
6.根据权利要求4所述的应用，其特征在于，所述基因编辑后的烟草植株盐胁迫后，其过氧化物酶含量和脯氨酸含量均高于对照烟草植株。
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CN113046371A (zh) * 2021-03-22 2021-06-29 云南中烟工业有限责任公司 一种烟草过氧化物酶相关的基因及其应用

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