CN114317558A - 一种日香桂冷敏感相关OfHSF11基因及其编码蛋白和应用 - Google Patents
一种日香桂冷敏感相关OfHSF11基因及其编码蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种日香桂冷敏感相关OfHSF11基因及其编码蛋白和应用,属于植物分子生物学领域。本发明基于植物基因克隆技术从日香桂中分离、克隆出的一个转录因子,命名为OfHSF11,其核苷酸序列如SEQ ID NO.1所示,在此基础上构建了超表达载体并进行本氏烟草的瞬时转化,通过在低温处理前后的表型分析,结果发现,转OfHSF11的植株冷敏感性明显高于非转OfHSF11的植株的冷敏感性,表明OfHSF11基因是一个潜在的抗寒性指示基因,为桂花抗寒遗传改良育种提供了有用的基因资源。
Description
技术领域
本发明属于植物分子生物学领域,具体涉及一种日香桂冷敏感相关OfHSF11基因及其编码蛋白和应用。
背景技术
桂花(Osmanthus fragrans),是我国长江以南地区园林绿化中广泛应用的一种常绿木本树种,观赏与经济价值较高,然而对低温胁迫敏感,在高纬度地区的栽培与应用受到限制。因此,在桂花中发掘抗寒基因,对于桂花抗寒性的提升具有重要的意义。
热休克转录因子(Heat shock transcription factor,HSF)作为植物热胁迫响应的关键组分之一,在植物热胁迫记忆中发挥重要作用。现有研究表明,HSF转录因子与植物的胁迫应答有关,但桂花中的HSF转录因子尚未被探索。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种从日香桂中分离、克隆得到的冷敏感相关调控因子OfHSF11;本发明所要解决的另一技术问题在于提供OfHSF11基因在桂花抗寒遗传改良育种及增强桂花冷敏感性中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种日香桂冷敏感相关OfHSF11基因,其核苷酸序列如SEQ ID NO.1所示,或者为编码SEQ ID NO.2的DNA序列。
所述的日香桂冷敏感相关OfHSF11基因编码的蛋白质,其氨基酸序列如SEQ IDNO.2示。利用ExPASy软件分析,发现该基因长度为1002bp,编码333个氨基酸蛋白,该蛋白的分子量为36.54kDa,等电点为5.24。
在SEQ ID NO.2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质,也属于本发明的保护范围。
含有所述的日香桂冷敏感相关OfHSF11基因的重组表达载体或重组菌株。
本申请构建了Super1300-OfHSF11的重组表达载体,并用其转化农杆菌GV3101。
所述的OfHSF11基因在增强植物冷敏感性中的应用,具体包括以下步骤:
1)构建含有如SEQ ID NO.1所示的OfHSF11基因的重组表达载体;
2)将步骤1)得到的重组表达载体转化农杆菌GV3101,得到重组农杆菌;
3)用重组农杆菌侵染本氏烟草,获得转基因植株,将转基因植株与未转基因植株进行冷胁迫处理,并进行温度耐受性相关基因的检测。
瞬时转基因苗冷胁迫处理:将瞬时注射后的本氏烟草放至培养箱,设定温度为4℃,湿度60%,光照250umol m-2s-1,处理6h后用无菌剪刀剪下注射过菌液的本氏烟草叶片,迅速放入液氮中冷冻样品,并移至-80℃超低温冰箱进行保存。
所述的日香桂冷敏感相关OfHSF11基因在桂花育种中的应用。
相比于现有技术,本发明的有益效果为:
本发明基于植物基因克隆技术从日香桂中分离、克隆出的一个转录因子,命名为OfHSF11,其核苷酸序列如SEQ ID NO.1所示,在此基础上构建了超表达载体并进行本氏烟草的瞬时转化,通过在低温处理前后的表型分析,结果发现,转OfHSF11的植株冷敏感性明显高于非转OfHSF11的植株的冷敏感性,表明OfHSF11基因是一个潜在的抗寒性指示基因,为桂花抗寒遗传改良育种提供了有用的基因资源。
附图说明
图1为目的基因OfHSF11扩增产物琼脂糖凝胶电泳图;
图2为目的基因OfHSF11扩增片段与Super1300载体连接转化后阳性单菌落检测图;
图3为双酶切后的载体琼脂糖凝胶电泳图;
图4为转化农杆菌GV3101后的菌检琼脂糖凝胶电泳图;
图5为瞬时转基因苗的半定量检测图;图中,泳道L1-L3为对照组瞬时转化植株,泳道LA-L6为目的基因瞬时转化植株的条带;Nbactin为内参基因;
图6为植物冷处理后的MDA含量测定图;图中,EV为空载植株,OfHSF11为OfHSF11瞬时转化植株;
图7为植物冷处理后抗寒相关基因表达量图;图中,EV为空载植株,OfHSF11为OfHSF11瞬时转化植株;NbAPX、NbSOD、NbCAT这三个为ROS清除相关基因;NbDREB3为冷反应调节基因;NbLEA5为冷应激防御蛋白的编码基因;NbHSP70、NbHSP90这2个为热激蛋白基因。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。以下实施例中未作具体说明的分子生物学实验方法,均可参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的方法或本领域的常规方法进行,或者按照试剂盒和产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本实施例采用TIANGEN植物RNA提取试剂盒(DP432)提取植物总RNA。采用TaKaRaPrimeScriptTMRT Master Mix(Perfect Rea1 Time)反转录试剂盒将所提取到的RNA反转录成cDNA,最后得到的cDNA加水稀释10倍后于-20℃冰箱保存。
实施例1构建日香桂OfHSF11基因的超表达载体
(1)获取目的基因
根据南京林业大学王良桂课题组已发表的桂花全基因组数据库(http:// 117.78.20.255/),筛选得到HSF基因家族的全部成员,并把其中的1个基因序列命名为OfHSF11。
(2)设计引物
利用BioXM软件对该基因的核苷酸全长序列进行酶切位点分析,选择Sma I和SpeI酶作为两个限制性内切酶。利用CE design软件设计引物。按要求进行相关信息的填写,具体包括载体上的酶切位点附近的序列、目的基因全长、按顺序填写2个酶切位点(5′端和3′端),即可得到扩增引物。设计好的序列由捷瑞生物公司合成,具体如下:
OfHSF11-F:5′-aagcttctgcaggggcccgggTAGGTGGGTCCGGCTATGG-3′;
OfHSF11-R:5′-catggtaccggatccactagtATTAAAATACCCTTGATTTCGCG-3′;
(3)载体双酶切
提前将Super1300载体从-80℃超低温冰箱中取出进行活化和摇菌,按照质粒提取试剂盒(北京天根生化科技有限公司)提取Super1300载体质粒,随后进行双酶切实验,体系(总共20μL体系)如下:Sma I酶1μL;Spe I酶1μL;Buffer 2μL;载体质粒XμL;ddH2O6μL。
其中X(μL)=1000ng/载体质粒浓度(ng/μL)。将离心管微微震荡,使其混匀,瞬时离心6s,置于37℃的水浴锅内培养1h。将获得的双酶切后的载体进行琼脂糖凝胶电泳,然后利用凝胶回收试剂盒(北京全式金生物技术有限公司)进行切胶回收。
(4)目的基因扩增
以稀释10倍的cDNA为模板,进行PCR扩增,体系如下:
OfHSF11-F1μL;OfHSF11-R 1μL;cDNA 1μL;Prime STAR 10μL;ddH2O7μL;总共20μL体系(Prime STAR即高保真PCR酶购买于宝日医生物技术(北京)有限公司高速高保真PCR酶R045A)。
每个基因做3个20μL体系。反应条件为:98℃变性10s;58℃退火15s;72℃延伸1min,35个循环;72℃总延伸10min;16℃终止反应。将获得的扩增产物进行琼脂糖电泳(图1),然后利用凝胶回收试剂盒(北京全式金生物技术有限公司)进行切胶回收。
(5)连接转化
连接体系如下:线性化载体XμL;插入目的片段YμL;5×CE II Buffer 4μL;ExnaseII 2μL(连接酶和Buffer源于南京诺唯赞生物科技有限公司同源重组试剂c112);ddH2OAdd to 20μL。
其中X=0.02×载体碱基对数(ng)/切胶回收线性载体浓度(ng/μL);Y=0.04×载体碱基对数(ng)/切胶回收目的片段浓度(ng/μL)。
将离心管微微震荡,使其混匀,瞬时离心6s,置于37℃的水浴锅内培养30min,冰上2min。
转化:超净工作台内,用移液枪取5μL的连接产物于50μL的TreliefTM 5α感受态细胞,轻弹混匀,冰浴5min,42℃水浴60s,再冰浴2min,加250μL液体LB(不含Kana),37℃,200rppm的摇床内孵育30min。
涂板:取200μL孵育后的菌液,用灭菌的玻璃棒均匀涂布在LB固体培养基上(内含50mg/L的Kana)并晾干,用封口膜后倒置在37℃恒温培养箱内培养12-14h。
(6)阳性单菌落检测和测序
当培养基上长出菌后,于超净工作台内进行单菌落检测。每个基因挑取8个饱满的单菌落,依序依次在含Kana抗性的LB固体培养基上进行备份,并用无菌牙签将相应的单菌落沾取至以下体系进行菌检:Super1300-F 1μL;OfHSF11-R1μL;Green Mix 10μL(GreenMix购于南京诺唯赞生物科技有限公司),ddH2O 8μL。
Super1300-F:5′-AACGCTTTACAGCAAGAACGGAATG-3′;
OfHSF11-R:5′-catggtaccggatccactagtATTAAAATACCCTTGATTTCGCG-3′;
PCR反应条件为:94℃预变性3min;94℃变性30s;58℃退火30s;72℃延伸1min,35个循环;72℃总延伸10min;16℃终止反应。将获得的扩增产物进行琼脂糖电泳(图2),挑取3个正确的阳性菌落送测。测得OfHSF11基因的核苷酸序列如SEQ ID No.1所示,其表达蛋白的氨基酸序列如SEQ ID NO.2所示,选取碱基错配率最低的阳性单菌落提取质粒进行后续操作。
(7)双酶切验证
将测序得到的序列正确的质粒进行双酶切验证,体系如下:
限制性内切酶11μL;限制性内切酶21μL;Buffer 2μL;载体质粒XμL;ddH2O 6μL;总体系为20μL。
其中X(μL)=1000ng/载体质粒浓度(ng/μL)。将离心管微微震荡,使其混匀,瞬时离心6s,置于37℃的水浴锅内培养1h。将获得的双酶切后的载体进行琼脂糖电泳(图3),检测双酶切情况。
实施例2转化农杆菌GV3101
(1)将保存在-80℃超低温冰箱内的GV3101感受态取出放在冰上融化。每33μL感受态加入1μL质粒,吸打混匀后依次冰浴20min、液氨速冻5min、37℃水浴5min、冰浴5min;
(2)加500μL无抗性的LB液体培养基,28℃,200rppm摇床上培养1h;
(3)培养完成后,将菌液6000r,离心1min,弃去部分上清液,留100μL均匀涂布于LB固体培养基上(内含50mg/LKana),封口膜密封,倒置于28℃培养箱中培养40-48h;
(4)菌检和备份:菌检中的目的条带正确且亮度一致(图4),则将备份的板子中相对应的菌落挑入LB液体培养基(内含50mg/LKana)中摇菌,再将菌液和50%甘油按3:7的体积比保菌,液氮中速冻后保存在-80℃超低温冰箱内。
实施例3侵染本氏烟草并进行丙二醛(MDA)指标测定
(1)摇菌:将Super1300空载、P19辅助表达载体和已转入农杆菌的GFP::Super1300-OfHSF11目标基因融合表达载体的菌液于-80℃取出常温融化至冰水混合状态后插入冰中融化,按照300μL菌液加入30mL LB液体培养基中(含Kana 10μg·mL-1),28℃、200rpm避光振荡培养,直至菌液OD600=0.6-0.8之间;
(2)准备混合菌液:称取0.0196g的乙酰丁香酮(AS)粉末,使用二甲基亚砜助溶(在通风橱操作),再加入适量无菌水定容到100ml,获得母液,取母液15ml加入85ml的无菌水,从而获得150μmol·L-1的AS;称取0.2035g的MgCl2和0.2135g的2-(N-吗啉代)乙磺酸(MES)加入到配好的150μmol·L-1的AS溶液中;将菌液时进行离心(4℃、5000rpm、10min),弃上清,收集菌体,使用当天配制的缓冲液进行重悬,最后按照最佳比例混合(P19辅助载体:目的基因(V∶V)=5∶7),充分震荡混匀后活化3h;
(3)侵染:生长健壮的35-40d左右的本氏烟草提前2天控水,使用1mL一次性注射器将混合菌液注入本氏烟草叶片背面;
(4)培养:将注射过目的基因混合菌液的瞬时转化植株和注射过空载体菌液的瞬时转化植株浇透水,放于生长室中培养2天(日照:15/9h,144μmol·m-2·s-1);
(5)瞬时转基因苗冷胁迫处理:将瞬时注射后的本氏烟草放至培养箱,设定温度为4℃(湿度60%,光照250umol m-2s-1),处理6h后用无菌剪刀剪下注射过菌液的本氏烟草叶片,迅速放入液氮中冷冻样品,并移至-80℃超低温冰箱进行保存。
(6)MDA含量测定参照李合生硫代巴比妥酸比色法(TAB)比色法(李合生.植物生理生化实验原理和技术[M].北京:高等教育出版社.2000.261-263)。
计算公式:(nmol·g-1FW)=[6.45(D532-D600)-0.56D450]×1000×Vt/(VS×W)
式中:D523、D600、D450分别为相应波长下的光密度值;
Vt-提取液总体积(mL);
W-样品鲜重(g);
Vs-测定时吸取酶液体积(mL);
(7)瞬时转化植株低温处理后的MDA含量分析:将数据导入SPSS中对空载植株和OfHSF11瞬时转化植株中测定的MDA含量进行差异性统计分析(p<0.01),与对照植株相比,经OfHSF11瞬时转化的烟草叶片MDA含量显著升高(图6),说明过表达OfHSF11改变了瞬时转化烟草叶片的透膜性,瞬时转化烟草的耐寒性显著降低。
(8)瞬时转化植株的半定量验证:采用TIANGEN植物RNA提取试剂盒(DP432)提取植物总RNA。采用TaKaRa PrimeScriptTM RT Master Mix(Perfect Real Time)反转录试剂盒将所提取到的RNA反转录成cDNA,最后得到的cDNA加水稀释10倍后于取1μL作为模板,加入RTOfHSF11-F 1μL,RTOfHSF11-R 1μL,Green Mix 10μL,ddH2O7μL,配成20μL的体系进行半定量实验,半定量实验中PCR程序为:94℃预变性3min;94℃变性30s;58℃退火30s;72℃延伸45s,35个循环;72℃总延伸10min;16℃终止反应;将获得的PCR产物进行琼脂糖电泳。
RTOfHSF11-F:5′-GGACTCAGGGGAAGAACAGGT-3′;
RTOfHSF11-R:5′-CATAGTCCGCCGAAATACCTT-3′;
(9)抗寒相关基因表达检测
qRT-PCR实验按照TaKaRa生物有限公司的TB GreenTM Premix Ex TaqTM试剂盒说明书进行操作。每个样品设置3个生物学重复及3个技术重复,总反应体系及程序如下:
qRT-PCR反应体系如下:cDNA(稀释20倍)1μL,ddH2O 3μL,Nbactin-qF 0.4μL,Nbactin-qR 0.4μL,Dye 0.2μL,SYBR 5μL。
Nbactin-qF:5′-CCACGAGACCACATACAACTCT-3′;
Nbactin-qR:5′-CTATCGGCAATACCTGGGAACA-3′;
qRT-PCR反应程序为:第一阶段:95℃3min;第二阶段:95℃45s,60℃ 30s,95℃15s,共40个循环;第三个阶段:60℃ 1min,95℃15s。
在尽量避开光线直射的条件下配好qRT-PCR的反应体系。体系配好后,用大离心机3000rpm/min 4℃条件下离心5min,使混合液全部聚集于反应连板的底部,然后设定qRT-PCR程序进行实验。Excel软件用来进行相对表达量分析,并用SPSS中的独立样本T检验比较显著性(*P<0.05,**P<0.01,***P<0.001)。
在过表达OfHSF11的烟草中,应激相关基因,如活性氧(reactive oxygenspecies,ROS)相关基因NbCAT、冷反应调节基因NbDREB3和冷应激防御蛋白编码基因NbLEA5的转录水平显著降低(图7)。这些结果表明,OfHSF11过表达植株的ROS清除系统可能受到干扰,从而增加了植株对冷胁迫条件的敏感性。
序列表
<110> 南京林业大学
<120> 一种日香桂冷敏感相关OfHSF11基因及其编码蛋白和应用
<130> 1
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1002
<212> DNA
<213> OfHSF11基因(Artificial)
<400> 1
atggctccgc caccggggga tccaaacgga gtaggggaat cgaccgccgg cgacgcgtca 60
aggacattac ctacaccgtt tctaacgaaa acttaccagc ttgtggatga ccacacaatt 120
gatgatgtta tttcatggaa tgaagatgga tctacattta tcgtatggaa tcctacggaa 180
ttcgctagag atttgctccc taaatatttc aagcacaaca atttctcaag ctttgtccgt 240
caactcaaca cttacggatt taggaaggtt gtgcctgatc ggtgggaatt ttcgaacgat 300
tgcttccggc gaggtgagaa aaagcttcta tgcgacatac agcgtcggaa aatagcaacg 360
atgtcttctg ccgtggcggc gacaccgtta actgcggtta cggtggcagt tgcaccttcg 420
acgccggctg caactcagca gctgacggta tctccttcgg actcagggga agaacaggtt 480
ctgtcatcct ccaactccgc tggggcgtgg tttggcggaa gcacatcgga gctgattgga 540
gagaacgagc gtctgaggaa agagaacatg cagctcaaca aagaattgag caacgtgaag 600
agcctatgca aaaatattta cgttctaatg tcaaatttcg ccaattgcag cagtagcaat 660
gaccaggcag taaagccgct ggatctgtta cctatgacaa gattttgtga agaaatgaaa 720
ggtgtcgtaa ccgccgccgc cgcggcgtct aacggcggcg gcgaagaaaa taacattaga 780
tctgaggcgg agagaataag cgcgaggctg ttcggggtgc cgataggcgc taaacgcggg 840
aaggaatgcg aaggtatttc ggcggactat gatatggatt tgcagctgca gcagcccggc 900
actgatgtga aatctgagcc ttctgatcaa aatagcggcg atgttcaaga gacaacctgg 960
cttaggaggt gcaaatcgcg aaatcaaggg tattttaatt ga 1002
<210> 2
<211> 333
<212> PRT
<213> OfHSF11基因编码的蛋白质(Artificial)
<400> 2
Met Ala Pro Pro Pro Gly Asp Pro Asn Gly Val Gly Glu Ser Thr Ala
1 5 10 15
Gly Asp Ala Ser Arg Thr Leu Pro Thr Pro Phe Leu Thr Lys Thr Tyr
20 25 30
Gln Leu Val Asp Asp His Thr Ile Asp Asp Val Ile Ser Trp Asn Glu
35 40 45
Asp Gly Ser Thr Phe Ile Val Trp Asn Pro Thr Glu Phe Ala Arg Asp
50 55 60
Leu Leu Pro Lys Tyr Phe Lys His Asn Asn Phe Ser Ser Phe Val Arg
65 70 75 80
Gln Leu Asn Thr Tyr Gly Phe Arg Lys Val Val Pro Asp Arg Trp Glu
85 90 95
Phe Ser Asn Asp Cys Phe Arg Arg Gly Glu Lys Lys Leu Leu Cys Asp
100 105 110
Ile Gln Arg Arg Lys Ile Ala Thr Met Ser Ser Ala Val Ala Ala Thr
115 120 125
Pro Leu Thr Ala Val Thr Val Ala Val Ala Pro Ser Thr Pro Ala Ala
130 135 140
Thr Gln Gln Leu Thr Val Ser Pro Ser Asp Ser Gly Glu Glu Gln Val
145 150 155 160
Leu Ser Ser Ser Asn Ser Ala Gly Ala Trp Phe Gly Gly Ser Thr Ser
165 170 175
Glu Leu Ile Gly Glu Asn Glu Arg Leu Arg Lys Glu Asn Met Gln Leu
180 185 190
Asn Lys Glu Leu Ser Asn Val Lys Ser Leu Cys Lys Asn Ile Tyr Val
195 200 205
Leu Met Ser Asn Phe Ala Asn Cys Ser Ser Ser Asn Asp Gln Ala Val
210 215 220
Lys Pro Leu Asp Leu Leu Pro Met Thr Arg Phe Cys Glu Glu Met Lys
225 230 235 240
Gly Val Val Thr Ala Ala Ala Ala Ala Ser Asn Gly Gly Gly Glu Glu
245 250 255
Asn Asn Ile Arg Ser Glu Ala Glu Arg Ile Ser Ala Arg Leu Phe Gly
260 265 270
Val Pro Ile Gly Ala Lys Arg Gly Lys Glu Cys Glu Gly Ile Ser Ala
275 280 285
Asp Tyr Asp Met Asp Leu Gln Leu Gln Gln Pro Gly Thr Asp Val Lys
290 295 300
Ser Glu Pro Ser Asp Gln Asn Ser Gly Asp Val Gln Glu Thr Thr Trp
305 310 315 320
Leu Arg Arg Cys Lys Ser Arg Asn Gln Gly Tyr Phe Asn
325 330
<210> 3
<211> 40
<212> DNA
<213> OfHSF11-F(Artificial)
<400> 3
aagcttctgc aggggcccgg gtaggtgggt ccggctatgg 40
<210> 4
<211> 44
<212> DNA
<213> OfHSF11-R(Artificial)
<400> 4
catggtaccg gatccactag tattaaaata cccttgattt cgcg 44
<210> 5
<211> 25
<212> DNA
<213> Super1300-F(Artificial)
<400> 5
aacgctttac agcaagaacg gaatg 25
<210> 6
<211> 21
<212> DNA
<213> RTOfHSF11-F(Artificial)
<400> 6
ggactcaggg gaagaacagg t 21
<210> 7
<211> 21
<212> DNA
<213> RTOfHSF11-R(Artificial)
<400> 7
catagtccgc cgaaatacct t 21
<210> 8
<211> 22
<212> DNA
<213> Nbactin-qF(Artificial)
<400> 8
ccacgagacc acatacaact ct 22
<210> 9
<211> 22
<212> DNA
<213> Nbactin-qR(Artificial)
<400> 9
ctatcggcaa tacctgggaa ca 22
Claims (8)
1.一种日香桂冷敏感相关OfHSF11基因,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的日香桂冷敏感相关OfHSF11基因编码的蛋白质,其特征在于,所述蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.含有权利要求1所述的日香桂冷敏感相关OfHSF11基因的重组表达载体或重组菌株。
4.权利要求1所述的OfHSF11基因在增强植物冷敏感性中的应用。
5.根据权利要求4所述的应用,其特征在于,包括以下步骤:
1)构建含有如SEQ ID NO.1所示的OfHSF11基因的重组表达载体;
2)将步骤1)得到的重组表达载体转化农杆菌,得到重组农杆菌;
3)用重组农杆菌侵染植物,并进行温度耐受性相关基因的检测。
6.根据权利要求5所述的应用,其特征在于,所述农杆菌为农杆菌GV3101。
7.根据权利要求5所述的应用,其特征在于,所述植物为本氏烟草。
8.权利要求1所述的日香桂冷敏感相关OfHSF11基因在桂花育种中的应用。
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CN115058435A (zh) * | 2022-06-24 | 2022-09-16 | 中国林业科学研究院华北林业实验中心 | 一种仁用杏Pasdehydrin-3基因及其在抗寒、促开花或种子结实中的应用 |
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CN114891078A (zh) * | 2022-04-13 | 2022-08-12 | 南京林业大学 | 一种日香桂抗盐相关OfNAC59基因及其编码蛋白和应用 |
CN115058435A (zh) * | 2022-06-24 | 2022-09-16 | 中国林业科学研究院华北林业实验中心 | 一种仁用杏Pasdehydrin-3基因及其在抗寒、促开花或种子结实中的应用 |
CN115058435B (zh) * | 2022-06-24 | 2023-05-05 | 中国林业科学研究院华北林业实验中心 | 一种仁用杏Pasdehydrin-3基因及其在抗寒、促开花或种子结实中的应用 |
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