CN111154774B - 葡萄VyLhcb4基因及其编码蛋白和基因在抗逆品种育种中的应用 - Google Patents
葡萄VyLhcb4基因及其编码蛋白和基因在抗逆品种育种中的应用 Download PDFInfo
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- CN111154774B CN111154774B CN202010187022.9A CN202010187022A CN111154774B CN 111154774 B CN111154774 B CN 111154774B CN 202010187022 A CN202010187022 A CN 202010187022A CN 111154774 B CN111154774 B CN 111154774B
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Abstract
本发明涉及葡萄VyLhcb4基因及其编码蛋白和基因在抗逆品种育种中的应用，属于植物基因工程技术领域。本发明中利用强启动子（花椰菜花叶病毒35S启动子）驱动原理的转基因技术，将燕山葡萄VyLhcb4基因的超量表达载体转入拟南芥中，从而获得转基因拟南芥植株；实验证明，相对于转化空载体的拟南芥植株，超量表达VyLhcb4基因导致转基因拟南芥中抗逆相关物质的积累和抗逆相关基因的表达，转基因植株抗逆性增强。因此，葡萄VyLhcb4基因及其重组表达载体能够用于植物抗逆品种育种。
Description
技术领域
本发明涉及植物基因工程技术领域，尤其涉及葡萄VyLhcb4基因及其编码蛋白和基因在抗逆品种育种中的应用。
背景技术
捕光叶绿素a/b结合蛋白是光合系统中的重要功能蛋白，定位于叶绿体类囊体膜上，与色素结合形成捕光色素蛋白复合体。该复合体能捕获光能，并迅速把光能传到PSI和光系统II的反应中心，引起光化学反应，将光能转化为化学能。在高等植物和绿藻中，PSI复合物由PSI核心复合物和捕光色素蛋白复合体I两部分组成。真核生物的PSI复合物包括了一系列捕光叶绿素a/b结合蛋白即LHCI，它们结合大约100个叶绿素分子以增大捕光截面，并且因为含有叶绿素b和类胡萝卜素而可以捕获光谱中不同波长的光。与PSII相比，PSI在植物上的研究较少，主要是以菠菜、豌豆和大麦等植物为试验材料，研究其蛋白组成、基因结构和结构生物学。PSI相对于PSII对逆境胁迫抵抗性强，是某些环境胁迫的最初作用部位。作为PSI中最重要的功能蛋白之一，LHCI蛋白目前的研究主要集中在光能的捕获与传递方面，涉及物种较少，而且基因结构、编码蛋白的生化特性、进化关系及胁迫应答等方面的研究报道尚少。通过对LHCI蛋白的研究有望发现与葡萄生长发育及抵御非生物胁迫相关的基因，将为葡萄抗逆性遗传改良提供新的思路。
发明内容
本发明的目的在于提供一种葡萄VyLhcb4基因，其能够增加转基因植株中抗逆相关物质的积累和逆性相关基因的表达，促进转基因植株逆性增强。该基因编码的葡萄VyLhcb4蛋白能够促进转基因植株中积累抗逆相关物质导致转基因植株逆性性增强。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种葡萄基因VyLhcb4，所述葡萄基因VyLhcb4的核苷酸序列如SEDID NO.1所示。
本发明还提供一种葡萄基因VyLhcb4编码蛋白，所述编码蛋白的氨基酸序列如SEDID NO.2所示。
本发明还提供了所述葡萄基因VyLhcb4在植物逆性品种育种中的应用。
作为优选，上述葡萄基因VyLhcb4应用于拟南芥或葡萄逆性品种育种中。
本发明中利用强启动子(花椰菜花叶病毒35S启动子)驱动原理的转基因技术，将燕山葡萄VyLhcb4基因的超量表达载体转入拟南芥中，从而获得转基因拟南芥植株；实验证明，相对于转化空载体的拟南芥植株，超量表达VyLhcb4基因导致转基因拟南芥中抗逆相关物质的积累和逆性相关基因的表达，转基因植株抗逆性增强。因此，葡萄VyLhcb4基因及其重组表达载体能够用于植物逆性品种育种。
附图说明
图1为本发明中葡萄VyLhcb4基因表达分析图；
图2为本发明中转VyLhcb4基因拟南芥植株的抗旱性鉴定图；
图3为本发明中转VyLhcb4基因拟南芥植株的生理特性分析图；
图4为本发明中转基因拟南芥植株中逆性相关基因的表达分析图。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1葡萄VyLhcb4基因表达分析
燕山葡萄组培苗继代培养16d后，选择生长健壮表现一致的幼苗用于各种逆境处理。
干旱处理：将葡萄幼苗从培养基中拔出，置于滤纸上暴露在室温为(32±1)℃、相对湿度为55％、光周期为光照14h/黑暗10h的条件下处理，在0、2、6、12、24h取样。
低温处理：将组培苗置于温度为(4±1)℃、相对湿度为75％、光周期为光照14h/黑暗10h的条件下培养，在0、2、6、12、24h取样。
盐胁迫：在三角瓶中加入20mL 100mmol·L-1的NaCl溶液，在温度为(25±1)℃、相对湿度为75％、光周期为光照14h/黑暗10h的条件下培养，在0、2、6、12、24h取样。
在三角瓶中加入等体积的蒸馏水作为盐胁迫处理的对照。正常培养的组培苗作为干旱和低温处理的对照。
在大田生长8～10年的燕山葡萄，在转色期取葡萄果实，于盛花期取根系(第一新生侧根)、茎(新展开叶下第4～5片叶的茎段)、叶(新展开叶下第4～5片)、花序和卷须(新生枝条的第1个枝)等样品。用plus植物总RNA提取试剂盒(天根)提取葡萄叶片总RNA。普通反转录用PrimeScript II 1st Strand cDNA Synthesis Kit(TaKaRa)合成cDNA第一链。
具体操作步骤如下：在PCR管中加入：Random 6mers(50μM)1μL，dNTP Mixture(10mM each)1μL，Total RNA 2μg，RNase free dH2O补齐至10μL，充分混匀，瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min，冰上急冷。
根据VyLhcb4基因序列设计实时荧光定量PCR引物，
正向引物序列为qRT-PCR-VyLhcb4-F：
5'-ACTCTCTCCAACATCTCCACACC-3'(如SEQ ID NO.3所示)；
反向引物序列为qRT-PCR-VyLhcb4-R：
5'-CTTTCTTACGACCGAACCCG-3'(如SEQ ID NO.4所示)。
以VyGAPDH基因为内参，
正向引物序列为qRT-VyGAPDH-F：
5'-cccttgtcctcccaactct-3'(如SEQ ID NO.5所示)；
反向引物序列为qRT-VyGAPDH-R：
5'-ccttctcagcactgtccct-3'(如SEQ ID NO.6所示)。
实时荧光定量PCR按照TaKaRa
Premix Ex TaqTMII(Perfect Real Time)说明在Bio-Rad IQ5 Real-Time PCR Detection System(Bio-Rad Laboratories,HercμLes，CA)上进行。25μL的反应体系：1μL的反转录模板；正反向引物各1μL；12.5μL的
Premix Ex TaqTM(2×)；9μL的nuclease-free water。反应程序为：95℃，30s；40cycles of95℃for 5s；57℃for 30s；72℃for 30s。结果采用2-ΔΔC(t)法进行分析。
结果如图1所示，结果表明VyLhcb4基因主要在根系中表达，其次在叶里表达量较高，在茎、花、果、卷须中的表达量较低；在低温处理后6h，VyLhcb4转录本快速积累，并到峰值，随后有所降低；在干旱处理后2h，VyLhcb4转录本快速积累，12h达到峰值，随后均有所降低；在高盐处理后2h，VyLhcb4转录本快速积累，24h并达到峰值。
试验例2转基因植株的获得
将包含有VyLhcb4基因编码区的ORF片段插入植物过量表达载体并将其转化入农杆菌中。将含有重组植物表达载体的农杆菌划线培养在LB平板(含60mg/L的Gent，100mg/L的Kan)上划线，置于28℃条件下培养24h；挑取单克隆在10mL LB液体培养基(附加相应的抗生素)中，在28℃条件下培养24h；取5mL菌液转移至50mL新鲜的LB液体培养基中，在28℃条件下继续培养，至菌液OD600达到0.6左右；转移至离心瓶或离心管中，室温条件下，转速为4000rpm离心10min，去除上清液收集菌体；重悬于渗透缓冲液(0.5×MS,5％蔗糖，0.03％Silwet L-77(GE Health))，调OD600至0.8；将拟南芥花序上已有的果荚去掉，花序完全浸入渗透液中10-30s(或用移液器直接将渗透液滴在花序上)，立即去掉拟南芥叶或茎秆上的渗透液，将植株平放在托盘中，用塑料薄膜覆盖托盘，24h后取下薄膜，于温室中继续培养；为提高转化效率，7天之后用同样方法再次侵染；经过转化的拟南芥植株进行正常管理，待果荚现白色时进行收种子。对上述植株通过卡那霉素进行筛选，得到的VyLhcb4转基因植株及转化空载体植株。对转基因植物和空载体植株分组并命名，其中空载体组命名为EV，转VyLhcb4基因拟南芥植株分为3组，分别命名为OE#1、OE#2、OE#3。
试验例3转基因拟南芥植株的抗旱性鉴定
VyLhcb4转基因植株和转化空载体的拟南芥(EV表示)在MS培养基上生长7天后，转移至营养钵中，正常浇水20天使其生长成健壮的幼苗。然后停止给拟南芥幼苗浇水即进行干旱处理，直到第7天部分拟南芥植株叶片出现明显的失水萎焉症状。之后对所有植株进行复水，48小时后观察植株生长状况。
干旱处理前后及复水后拟南芥植株的表现型通过拍照记录，结果如图2所示，从图2中可以看出与转化空载体拟南芥相比，转VyLhcb4基因拟南芥植株OE#1、OE#2、OE#3的抗旱能力明显增强。
试验例4转基因拟南芥植株生理生化特性分析
失水率的测定：VyLhcb4转基因植株和转化空载体植株正常生长3周后，分别取约0.2g的莲座叶进行失水率的测定。将采取的莲座叶放置于干燥的滤纸上，每隔10min测量一次叶片的鲜重(FW)，直到测至50min时失水率测定结束。将每一次测定的失水量与第一次测定的鲜重的比值作为失水率。
电解质渗漏率(电导率)的测定：将叶片装入离心管中，用超去离子水定容至10mL，室温下振荡1小时后测定溶液的电导值，记为煮前C1。随后将溶液连同叶片置于沸水中煮沸10min后，等温度降至室温后测定电导值，记为C2。将C1与C2的比值(C1/C2)作为相对电解质渗漏值。
检测结果如图3所示，(A)转基因拟南芥植株中VyLhcb4基因的表达量检测；(B)VyLhcb4基因拟南芥植株在干旱处理18d后的存活率统计；(C)转VyLhcb4基因拟南芥叶片相对失水率；(D)转VyLhcb4基因拟南芥植株在干旱处理18d后的相对电导率。从图中可以看出，转基因拟南芥植株中VyLhcb4基因表达量较高，存活率相对于转化空载体的植株显著提高；相对于转化空载体植株，转VyLhcb4基因拟南芥植株中失水率和电导率显著降低。
试验例5转基因拟南芥抗旱相关基因表达分析
用plus植物总RNA提取试剂盒提取干旱处理后的转基因拟南芥叶片总RNA。普通反转录用PrimeScriptII 1st Strand cDNA Synthesis Kit(TaKaRa)合成cDNA第一链。具体操作步骤如下：在PCR管中加入：Random 6mers(50μM)1μL，dNTP Mixture(10mM each)1μL，Total RNA2μg，RNase free dH2O补齐至10μL，充分混匀，瞬时离心使溶液至PCR管底部。在PCR仪上65℃反应5min，冰上急冷。以拟南芥AtActin为内参基因，检测转基因拟南芥植株中抗旱相关基因AtCOR15A、AtERD15、AtRD29A、AtP5CS1基因的表达。所设计引物如下所示：
qRT-AtActin-F：5’-CGGTGGTTCTATCTTGGCATC-3’(如SEQ ID NO.7所示)；
qRT-AtActin-R：5’-GTCTTTCGCTTCAATAACCCTA-3’(如SEQ ID NO.8所示)；
qRT-AtCOR15A-F：5’-CAGCGGAGCCAAGCAGAGCAG-3’(如SEQ ID NO.9所示)；
qRT-AtCOR15A-R：5’-CATCGAGGATGTTGCCGTCACC-3’(如SEQ ID NO.10所示)；
qRT-AtERD15-F：5’-CCAGCGAAATGGGGAAACCA-3’(如SEQ ID NO.11所示)；
qRT-AtERD15-R：5’-ACAAAGGTACAGTGGTGGC-3’(如SEQ ID NO.12所示)；
qRT-AtRD29A-F：5’-GTTACTGATCCCACCAAAGAAGA-3’(如SEQ ID NO.13所示)；
qRT-AtRD29A-R：5’-GGAGACTCATCAGTCACTTCCA-3’(如SEQ ID NO.14所示)；
qRT-AtP5CS1-F：5’-CGACGGAGACAATGGAATTGT-3’(如SEQ ID NO.15所示)；
qRT-AtP5CS1-R：5’-GATCAGAAATGTGTAGGTAGC-3’(如SEQ ID NO.16所示)。
实时荧光定量PCR按照TaKaRa
Premix Ex TaqTMII(Perfect RealTime)说明在Bio-Rad IQ5 Real-Time PCR Detection System(Bio-Rad Laboratories,HercμLes,CA)上进行。25μL的反应体系：1μL的反转录模板；正反向引物各1μL；12.5μL的
Premix Ex TaqTM(2×)；9μL的nuclease-free water。反应程序为：95℃，30s；40cyclesof95℃for 5s；57℃for 30s；72℃for 30s。结果采用2-ΔΔC(t)法进行分析。
检测结果如图4所示，从图4中可以看出，在干旱条件下，与转化空载体拟南芥相比，转基因拟南芥植株中抗逆相关基因表达量明显升高，表明本发明中的野葡萄VyLhcb4基因，能够增加转基因植株中抗逆相关物质的积累和抗逆相关基因的表达，促进转基因植株抗逆性增强。
由以上实施例可知，本发明提供了一种葡萄基因VyLhcb4及其在抗逆育种中的应用。本发明中利用强启动子(花椰菜花叶病毒35S启动子)驱动原理的转基因技术，将燕山葡萄VyLhcb4基因的超量表达载体转入拟南芥中，从而获得转基因拟南芥植株；实验证明，相对于转化空载体的拟南芥植株，超量表达VyLhcb4基因导致转基因拟南芥中抗逆相关物质的积累和抗逆相关基因的表达，转基因植株抗逆性增强。因此，葡萄VyLhcb4基因及其重组表达载体能够用于植物抗逆品种育种。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 河南科技大学
<120> 葡萄VyLhcb4基因及其编码蛋白和基因在抗旱品种育种中的应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1075
<212> DNA
<213> VyLhcb4
<400> 1
tcgaatcctc acaccaccac aattccattc tcactctctc caacatctcc acacccgcca 60
ctccttacac aacagccatg gcaaccacca ctgccgccgc cgccgccgct acgtcctcat 120
tcatgggaac acgccttccc gacgtccact caaacaccgg ccgcgtccaa gcccgattcg 180
ggttcggtcg taagaaagcg cccgcaaaga aggccgcgaa gcgggtgtcc gatcgcccgc 240
tatggttccc gggagccgta gctccagagt ggctggatgg gaccttggtt ggggactacg 300
gatttgaccc cttcggtttg ggcaagcccg ccgagtactt gcagtacgat tacgactcct 360
tggaccagaa cttggccaag aacatcgccg gagacgtcat cggaaccagg ttcgagggcg 420
gcgaggtgaa gtcgaccccg ttccagcctt acaccgaggt ttttgggctg cagcggttcc 480
gtgagtgcga gctcattcat gggaggtggg ccatgttggc cacactcggt gctctcgccg 540
tggagtcact caccggagtc acatggcaag atgccggaaa ggtggagctg atcgaaggat 600
catcgtatct agggcaacca cttccattct ccatgaccac attgatctgg attgaagttc 660
tggtgattgg gtacattgag ttccagagga atgcagagct ggacccagaa aagaggctgt 720
acccaggtgg aaagttcttc gacccattgg gattggcagc tgacccggag aagaaggcgg 780
tcctccaact ggcggagatc aagcacgctc gccttgccat gattggtttc ctgggattcg 840
ccgtccaggc agccgtcacc ggaaaaggcc ctctcaacaa ctgggccacc catttgagcg 900
acccacttca cacaaccatt atcgacaacg ttttctactc ttaagctctc ccagtatttc 960
tcccttgtgc caatatttta taatagctga gatgtcgtga ggtatctgta tttgccatct 1020
tgtaaagaac gtaacattca cccttaagca caacgctatt gccctctatt atcaa 1075
<210> 2
<211> 288
<212> PRT
<213> VyLhcb4
<400> 2
Met Ala Thr Thr Thr Ala Ala Ala Ala Ala Ala Thr Ser Ser Phe Met
1 5 10 15
Gly Thr Arg Leu Pro Asp Val His Ser Asn Thr Gly Arg Val Gln Ala
20 25 30
Arg Phe Gly Phe Gly Arg Lys Lys Ala Pro Ala Lys Lys Ala Ala Lys
35 40 45
Arg Val Ser Asp Arg Pro Leu Trp Phe Pro Gly Ala Val Ala Pro Glu
50 55 60
Trp Leu Asp Gly Thr Leu Val Gly Asp Tyr Gly Phe Asp Pro Phe Gly
65 70 75 80
Leu Gly Lys Pro Ala Glu Tyr Leu Gln Tyr Asp Tyr Asp Ser Leu Asp
85 90 95
Gln Asn Leu Ala Lys Asn Ile Ala Gly Asp Val Ile Gly Thr Arg Phe
100 105 110
Glu Gly Gly Glu Val Lys Ser Thr Pro Phe Gln Pro Tyr Thr Glu Val
115 120 125
Phe Gly Leu Gln Arg Phe Arg Glu Cys Glu Leu Ile His Gly Arg Trp
130 135 140
Ala Met Leu Ala Thr Leu Gly Ala Leu Ala Val Glu Ser Leu Thr Gly
145 150 155 160
Val Thr Trp Gln Asp Ala Gly Lys Val Glu Leu Ile Glu Gly Ser Ser
165 170 175
Tyr Leu Gly Gln Pro Leu Pro Phe Ser Met Thr Thr Leu Ile Trp Ile
180 185 190
Glu Val Leu Val Ile Gly Tyr Ile Glu Phe Gln Arg Asn Ala Glu Leu
195 200 205
Asp Pro Glu Lys Arg Leu Tyr Pro Gly Gly Lys Phe Phe Asp Pro Leu
210 215 220
Gly Leu Ala Ala Asp Pro Glu Lys Lys Ala Val Leu Gln Leu Ala Glu
225 230 235 240
Ile Lys His Ala Arg Leu Ala Met Ile Gly Phe Leu Gly Phe Ala Val
245 250 255
Gln Ala Ala Val Thr Gly Lys Gly Pro Leu Asn Asn Trp Ala Thr His
260 265 270
Leu Ser Asp Pro Leu His Thr Thr Ile Ile Asp Asn Val Phe Tyr Ser
275 280 285
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
actctctcca acatctccac acc 23
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctttcttacg accgaacccg 20
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cccttgtcct cccaactct 19
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccttctcagc actgtccct 19
<210> 7
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cggtggttct atcttggcat c 21
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtctttcgct tcaataaccc ta 22
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cagcggagcc aagcagagca g 21
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
catcgaggat gttgccgtca cc 22
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccagcgaaat ggggaaacca 20
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
acaaaggtac agtggtggc 19
<210> 13
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gttactgatc ccaccaaaga aga 23
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggagactcat cagtcacttc ca 22
<210> 15
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgacggagac aatggaattg t 21
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gatcagaaat gtgtaggtag c 21
Claims (2)
1.葡萄基因VyLhcb4在植物抗旱品种育种中的应用，其特征在于，所述葡萄基因VyLhcb4的核苷酸序列如SED ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述植物为拟南芥或葡萄。
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|---|---|---|---|---|
| CN1813060A (zh) * | 2003-04-15 | 2006-08-02 | 巴斯福植物科学有限公司 | 对环境胁迫具有提高的耐性的植物细胞和植物 |
| AU2007214296A1 (en) * | 1998-08-04 | 2007-09-20 | Cropdesign N.V. | Genes involved in tolerance to environmental stress |
| CN101115841A (zh) * | 2004-12-16 | 2008-01-30 | 塞瑞斯公司 | 用于增强植物干旱耐受性的核苷酸序列及其编码的多肽 |
| CN102229953A (zh) * | 2011-06-03 | 2011-11-02 | 清华大学 | 捕光色素叶绿素a/b结合蛋白LHCB4在植物育种中的应用 |
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| CN102229953A (zh) * | 2011-06-03 | 2011-11-02 | 清华大学 | 捕光色素叶绿素a/b结合蛋白LHCB4在植物育种中的应用 |
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