CN110885841B - 葡萄VyCYP89A2基因及其编码蛋白和基因在抗旱品种育种中的应用 - Google Patents
葡萄VyCYP89A2基因及其编码蛋白和基因在抗旱品种育种中的应用 Download PDFInfo
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Abstract
本发明涉及葡萄VyCYP89A2基因及其编码蛋白和基因在抗旱品种育种中的应用，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VyCYP89A2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VyCYP89A2基因导致转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达，转基因植株抗旱性增强。因此，葡萄VyCYP89A2基因及其重组表达载体能够用于植物抗旱品种育种。
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VyCYP89A2基因及其编码蛋白和基因在抗旱品种育种中的应用。
背景技术
葡萄原产亚洲西部，世界各地均有栽培，世界各地的葡萄约95％集中分布在北半球，中国主要产区有安徽的萧县，新疆的吐鲁番、和田，山东的烟台，河北的张家口、宣化、昌黎，辽宁的大连、熊岳、沈阳及河南的芦庙乡、民权、仪封等地。在葡萄生长发育过程中水分对于葡萄的产量和品质有重要的影响，缺水容易使葡萄产业受到很大的威胁。在水资源匮乏的大背景之下，发掘抗旱葡萄资源、研究葡萄抗旱基因对提高葡萄抗旱性、培育抗旱新品种及节水栽培等都具有重要的科学价值和意义。
CYP是CytochromeP450的缩写，细胞色素P450是一个古老的大家族，在有机体中普遍存在，随着越来越多植物基因组测序的完成，发现植物细胞色素P450约占植物基因组的1％。P450中的CYP86是一个新发现的家族，包含4个亚家族，该家族基因多编码脂肪酸羟化酶，参与脂肪酸的分解代谢，与植物生长发育和非生物胁迫的防御密切相关，但目前葡萄中CYP86家族基因功能的研究还未见报道，还需进一步探究，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VyCYP89A2基因，其能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达，促进转基因植株抗旱性增强。该基因编码的葡萄VyCYP89A2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抗旱性增强。
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VyCYP89A2，所述葡萄基因VyCYP89A2的核苷酸序列如SED ID NO.1所示。
本发明还提供一种葡萄基因VyCYP89A2编码蛋白，所述编码蛋白的氨基酸序列如SEDIDNO.2所示，该编码蛋白的核苷酸序列位于SEDIDNO.1中的第29～1594位。
本发明还提供了所述葡萄基因VyCYP89A2在植物抗旱品种育种中的应用。
作为优选，上述葡萄基因VyCYP89A2应用于拟南芥或葡萄抗旱品种育种中。
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VyCYP89A2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VyCYP89A2基因导致转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达，转基因植株抗旱性增强。因此，葡萄VyCYP89A2基因及其重组表达载体能够用于植物抗旱品种育种。
附图说明
图1为本发明中葡萄VyCYP89A2基因表达分析图；
图2为本发明中转VyCYP89A2基因拟南芥植株的抗旱性鉴定图；
图3为本发明中转VyCYP89A2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抗旱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VyCYP89A2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 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VyCYP89A2基因序列设计实时荧光定量PCR引物，
正向引物序列为qPCR-VyCYP89A2-F：
5'-AACACACACAGGACCAGAGACT-3'(如SEQ ID NO.3所示)；
反向引物序列为qPCR-VyCYP89A2-R：
5'-CCTCTAAGCCGAGAGACCAGA-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(PerfectReal 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VyCYP89A2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、干旱、高盐处理后2h，VyCYP89A2转录本快速积累，在6h达到峰值，随后逐渐降低。
试验例2转基因植株的获得
将包含有VyCYP89A2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VyCYP89A2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VyCYP89A2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
VyCYP89A2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VyCYP89A2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：VyCYP89A2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VyCYP89A2基因的表达量检测；(B)VyCYP89A2基因拟南芥植株在干旱处理18d后的存活率统计；(C)转VyCYP89A2基因拟南芥叶片相对失水率；(D)转VyCYP89A2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VyCYP89A2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VyCYP89A2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PrimeScriptII1st 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。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；40cycles of95℃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旱相关基因表达量明显升高，表明本发明中的野葡萄VyCYP89A2基因，能够增加转基因植株中抗逆相关物质的积累和抗旱相关基因的表达，促进转基因植株抗旱性增强。
由以上实施例可知，本发明提供了一种葡萄基因VyCYP89A2及其在抗旱育种中的应用。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VyCYP89A2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VyCYP89A2基因导致转基因拟南芥中抗逆相关物质的积累和抗旱相关基因的表达，转基因植株抗旱性增强。因此，葡萄VyCYP89A2基因及其重组表达载体能够用于植物抗旱品种育种。
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VyCYP89A2基因及其在抗旱品种育种中的应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1789
<212> DNA
<213> VyCYP89A2(葡萄)
<400> 1
aacacacaca ggaccagaga ctttttcaat ggagatatgg gtcttcctct tcgtcgtctc 60
cctctgcatt gcttctctcc tcaaatctct ccatgatttc ttcttcccaa agctcaatct 120
cccaccagga cccgctgcct ttcctctcat cggaaacctc cactggctcg gcccatcctt 180
cgccgacctc gaacccatac ttcgaaattt acacgccaag tacggcccaa tcctcactct 240
ccgaatcggc tctcgcccgg ccatcttcat ctccgagaac tccctcgccc accaggcctt 300
ggtgcaaaac ggagccgttt tcgccgaccg tcctgcggcg cttccggcca gtagagtcat 360
gagcagtaac cagcgaaaca tcaattcgag cccctatggc ccgacctggc gtctcctccg 420
ccgcaatctc acggccgaga ttctccactc ttcccgggtg agaagctact cccacgcacg 480
gaagtgggtg ctggagattc tggtctctcg gcttagaggc cactccgacg gatttgtccc 540
cgtccgaata atggatcact ttcaatacgc catgttttgc ctactggtgc tcatgtgttt 600
cggggacaag ctcgaggaga agcaaattca ggagattgag atgattcaac gaaagttgtt 660
gttggcgttc cgtggattga ataggctcaa tttgtggccg aggatgggga agatattatt 720
tcgaaaaagg tgggaggagt ggcttaattt gcgcaaggat caggaagcga ttcttctgcc 780
gcacataaga gccagacagc ggcttaagca ggaaacgcaa aacaaacaag aagatgattc 840
atcttcatct tcaaaggact atgtcttgtc gtatgtggac actttgctgg atctgcagct 900
gccggaggag aagaggaagc ttaatgaagg agagatggtg acgctgtgct cggagtttct 960
cagtgccgga actgacacca cctccactgc actgcagtgg atcatggcga acctggtgaa 1020
ggccccccat attcaggcca ggcttttcga ggagattagt ggggttgtgg gggagggaga 1080
ggaggaggtg aaggaggagg atttgcagaa gatgccatac ttgaaggcag tggtcttgga 1140
aggtctgagg cggcatcctc cgggacactt tgtgttgcct cattcagtga cccaagatgt 1200
aagctttgaa gggtatgaca tacccaagaa tgcaactgtg aatttctcag tatcagatat 1260
gaattggaac ccaacgattt gggaagatcc aatggagttc aagccagaga ggttcttgaa 1320
cagcaatgga gatggagatc atgcagatgc agggaaagaa tttgatataa ctgggagcaa 1380
agagataaag atgatgccat ttggtgcagg gaggaggatt tgtcctgggt atgggttggc 1440
aatgctgcat ttggagtatt ttgtggggaa tttggtgtgg aattttgaat ggaaagcagt 1500
ggagggagat gaagttgatc tatctgagaa gctggagttc acggtagtga tgaagaatcc 1560
attgcaggct cacttatctc caaggttgaa ataatttata aataaataaa aaaataaaga 1620
agtagacggc tttagaagac cactactact tcatcatcac cataattctt atgatatttc 1680
agtataatgt atggatttga actgcacatt ctataacata tatgcagaat attaaaggga 1740
aagttggctc gatgtgtatg caaaatcgta agaaagtggg ttatctttc 1789
<210> 2
<211> 521
<212> PRT
<213> VyCYP89A2(葡萄)
<400> 2
Met Glu Ile Trp Val Phe Leu Phe Val Val Ser Leu Cys Ile Ala Ser
1 5 10 15
Leu Leu Lys Ser Leu His Asp Phe Phe Phe Pro Lys Leu Asn Leu Pro
20 25 30
Pro Gly Pro Ala Ala Phe Pro Leu Ile Gly Asn Leu His Trp Leu Gly
35 40 45
Pro Ser Phe Ala Asp Leu Glu Pro Ile Leu Arg Asn Leu His Ala Lys
50 55 60
Tyr Gly Pro Ile Leu Thr Leu Arg Ile Gly Ser Arg Pro Ala Ile Phe
65 70 75 80
Ile Ser Glu Asn Ser Leu Ala His Gln Ala Leu Val Gln Asn Gly Ala
85 90 95
Val Phe Ala Asp Arg Pro Ala Ala Leu Pro Ala Ser Arg Val Met Ser
100 105 110
Ser Asn Gln Arg Asn Ile Asn Ser Ser Pro Tyr Gly Pro Thr Trp Arg
115 120 125
Leu Leu Arg Arg Asn Leu Thr Ala Glu Ile Leu His Ser Ser Arg Val
130 135 140
Arg Ser Tyr Ser His Ala Arg Lys Trp Val Leu Glu Ile Leu Val Ser
145 150 155 160
Arg Leu Arg Gly His Ser Asp Gly Phe Val Pro Val Arg Ile Met Asp
165 170 175
His Phe Gln Tyr Ala Met Phe Cys Leu Leu Val Leu Met Cys Phe Gly
180 185 190
Asp Lys Leu Glu Glu Lys Gln Ile Gln Glu Ile Glu Met Ile Gln Arg
195 200 205
Lys Leu Leu Leu Ala Phe Arg Gly Leu Asn Arg Leu Asn Leu Trp Pro
210 215 220
Arg Met Gly Lys Ile Leu Phe Arg Lys Arg Trp Glu Glu Trp Leu Asn
225 230 235 240
Leu Arg Lys Asp Gln Glu Ala Ile Leu Leu Pro His Ile Arg Ala Arg
245 250 255
Gln Arg Leu Lys Gln Glu Thr Gln Asn Lys Gln Glu Asp Asp Ser Ser
260 265 270
Ser Ser Ser Lys Asp Tyr Val Leu Ser Tyr Val Asp Thr Leu Leu Asp
275 280 285
Leu Gln Leu Pro Glu Glu Lys Arg Lys Leu Asn Glu Gly Glu Met Val
290 295 300
Thr Leu Cys Ser Glu Phe Leu Ser Ala Gly Thr Asp Thr Thr Ser Thr
305 310 315 320
Ala Leu Gln Trp Ile Met Ala Asn Leu Val Lys Ala Pro His Ile Gln
325 330 335
Ala Arg Leu Phe Glu Glu Ile Ser Gly Val Val Gly Glu Gly Glu Glu
340 345 350
Glu Val Lys Glu Glu Asp Leu Gln Lys Met Pro Tyr Leu Lys Ala Val
355 360 365
Val Leu Glu Gly Leu Arg Arg His Pro Pro Gly His Phe Val Leu Pro
370 375 380
His Ser Val Thr Gln Asp Val Ser Phe Glu Gly Tyr Asp Ile Pro Lys
385 390 395 400
Asn Ala Thr Val Asn Phe Ser Val Ser Asp Met Asn Trp Asn Pro Thr
405 410 415
Ile Trp Glu Asp Pro Met Glu Phe Lys Pro Glu Arg Phe Leu Asn Ser
420 425 430
Asn Gly Asp Gly Asp His Ala Asp Ala Gly Lys Glu Phe Asp Ile Thr
435 440 445
Gly Ser Lys Glu Ile Lys Met Met Pro Phe Gly Ala Gly Arg Arg Ile
450 455 460
Cys Pro Gly Tyr Gly Leu Ala Met Leu His Leu Glu Tyr Phe Val Gly
465 470 475 480
Asn Leu Val Trp Asn Phe Glu Trp Lys Ala Val Glu Gly Asp Glu Val
485 490 495
Asp Leu Ser Glu Lys Leu Glu Phe Thr Val Val Met Lys Asn Pro Leu
500 505 510
Gln Ala His Leu Ser Pro Arg Leu Lys
515 520
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agcagtaacc agcgaaacat ca 22
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cctctaagcc gagagaccag a 21
<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 (1)
1.葡萄基因VyCYP89A2在植物抗旱品种育种中的应用，所述植物抗旱品种为拟南芥或葡萄；
所述葡萄基因VyCYP89A2的核苷酸序列如SED ID NO.1所示。
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