CN106754956B - 棉花GhJAZ11-D基因鉴定及应用 - Google Patents
棉花GhJAZ11-D基因鉴定及应用 Download PDFInfo
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- CN106754956B CN106754956B CN201611081723.4A CN201611081723A CN106754956B CN 106754956 B CN106754956 B CN 106754956B CN 201611081723 A CN201611081723 A CN 201611081723A CN 106754956 B CN106754956 B CN 106754956B
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Abstract
本发明涉及一个棉花GhJAZ11‑D基因鉴定及应用。GhJAZ11‑D基因开放阅读框全长687bp，编码一个含有228氨基酸的蛋白质。该基因在棉纤维起始期高量表达，且在纤维伸长期显示出表达量逐渐上升的趋势。在棉花中过量表达GhJAZ11‑D基因导致转基因棉花纤维长度显著增加，纤维品质也有所提高，这表明该基因参与调控棉花纤维细胞的发育过程。GhJAZ11‑D蛋白定位于细胞核中，能够与茉莉素信号通路中的关键因子GhCOI1和GhMYC2发生相互作用，表明GhJAZ11‑D参与棉花茉莉素信号调控。上述结果表明，GhJAZ11‑D可能通过影响茉莉素信号通路来调节棉纤维发育及纤维品质性状。综上所述，本发明提供了一个在棉纤维品质改良中具有重要应用价值的功能基因。
Description
技术领域：
本发明涉及一个棉花基因GhJAZ11-D的克隆与功能鉴定。
背景技术：
棉花是世界上最重要的经济作物之一。我国是世界上最大的产棉国和棉花消费国，棉花生产在我国国民经济中占有十分重要的地位。棉纤维是棉花生产的主要产品，其产量和品质直接决定了棉花生产的产值和效益，因此，一直以来，提高棉纤维产量和品质都是棉花育种的主要目标。
棉花纤维是由胚珠外被上的表皮细胞分化而成的一种无分支的种子表皮毛。每个棉纤维细胞都是单细胞，且在其伸长过程中不具有细胞分裂和多细胞发育过程，因此，棉纤维细胞是研究细胞分化和伸长的理想材料(Kim and Triplett，2001)。棉纤维细胞分化和发育过程可分为纤维细胞起始(开花前3天–开花后5天)、快速伸长(开花当天–开花后20天)、次生壁沉积(开花后16–40天)和脱水成熟(开花后40–50天)四个不同而又重叠的时期(Basra and Malik，1984)。纤维细胞的起始与伸长直接影响到棉纤维的数量和长度，而次生壁加厚则影响棉纤维的强度和马克隆值等性状。因此，克隆棉花纤维发育相关基因，研究基因产物之间的关系，解析棉花纤维发育的分子机制，不仅具有重要的理论意义，而且对棉花纤维产量和品质改良具有重要的应用价值。
棉花纤维发育过程受到细胞内植物激素水平的调控。采用棉花离体胚珠培养技术，Beasley和Ting检测了不同激素对棉花纤维分化及发育过程中的作用，结果表明吲哚乙酸(IAA)和赤霉素(GA)可以促进棉花纤维的起始和伸长，而细胞分裂素和脱落酸(ABA)对纤维的发育有抑制作用(Beasley and Ting，1973)。后来的研究发现，油菜素内脂(BR)对纤维起始分化和伸长也是必需的(Sun et al.,2005)。乙烯可以通过促进蔗糖合成酶、微管蛋白和膨胀素相关基因的表达来促进纤维伸长(Shi et al.,2006)。近年来的研究还发现，外源的茉莉素(JA)可以抑制棉花纤维的伸长(Tan et al.,2012)。
茉莉素(JA)作为一种重要的植物激素，广泛分布于植物幼嫩组织、花和发育的生殖器官中(Browse，2009)，通过信号转导途径调控植物生长发育和对外界环境的防御应答。研究表明，JA在植物体内具有多种生理功能，概括起来有如下两大方面：(1)调节植物生长发育。例如，促进叶片衰老、抑制种子萌发、抑制幼苗生长和根的伸长，与雌蕊的发育、表皮毛发育和与花青素积累相关等。(2)作为信号分子参与各种生物和非生物胁迫应答(Qi etal.,2011；Wasternack，2007)。茉莉素信号通路有三个关键因子：受体COI1，抑制因子JAZ和下游主要正调控因子MYC2。当植物体内JA浓度较低时，JAZ与MYC2互作，从而抑制MYC2对下游JA应答基因的正调控作用。而当植物受到刺激或发育需要时，植物体内存在较高的JA浓度，JA的活化形式(JA-Ile)被受体COI1识别并结合，招募JAZ蛋白，形成COI1-JA-Ile-JAZ复合体，使JAZ被SCFCOI1的E3连接酶泛素化，并以依赖于26S蛋白酶体的形式被降解，从而使MYC2可以结合到下游JA应答基因的启动子上，促进下游基因表达，从而引发JA信号通路(Howe，2010)。
JA是拟南芥表皮毛发育的正调控因子，JA可以去除其抑制因子JAZ与调控表皮毛起始的三联复合体WD-Repeat/bHLH/MYB中的bHLH和MYB因子的互作，从而促进表皮毛的发生(Qi et al.,2011)。同时，JA与GA和IAA之间存在拮抗作用，抑制表皮毛细胞的伸长。在JA信号通路中，JAZ作为关键的抑制因子，发挥着重要的作用。目前，在拟南芥中，已经鉴定了13个家族成员，其蛋白序列中含有3个结构域：NT结构域、ZIM结构域和Jas结构域。其中，Jas结构域，含有22个氨基酸，位于JAZ蛋白的C端，序列高度保守，是JAZ蛋白区别于其他ZIM蛋白的主要特征，该结构域的主要作用是可以与其他转录因子及JA受体COI1蛋白发生相互作用(Kazan and Manners,2010；Thini et al.,2007)。因此，JAZ是JA信号通路中的一个关键负调控因子，以此为切入点，研究植物激素JA在棉花纤维起始和伸长的作用，对于棉花纤维品质改良，具有重要意义。
发明内容：
本发明的目的在于提供一个新的棉花JAZ基因GhJAZ11-D，分析揭示该基因的生物学功能，探索其对纤维发育调控的作用机制，进而应用该基因改良棉纤维品质。
在最近的研究中，我们通过公开的四倍体棉花基因组数据库鉴定了30个编码JAZ蛋白的基因，其中一个基因命名为GhJAZ11-D。该基因含有687bp开放阅读框，编码228个氨基酸的JAZ蛋白。GhJAZ11-D蛋白具有NT结构域、保守的ZIM结构域和Jas结构域。系统进化分析表明，GhJAZ11-D与AtJAZ11和AtJAZ12位于同一进化分支。利用荧光定量RT-PCR技术对该基因在棉花不同组织和棉纤维不同发育阶段的表达谱进行了分析，结果表明GhJAZ11-D基因在棉花不同组织以及不同的纤维发育阶段都有一定的表达，而且在纤维起始期高量表达，在之后的纤维伸长期其表达量逐渐上升(图1)。亚细胞定位分析表明，GhJAZ11-D蛋白定位于细胞核中(图2)。利用酵母双杂交技术验证GhJAZ11-D与受体GhCOI1蛋白的相互作用，结果显示GhJAZ11-D与GhCOI1结合后形成的二倍体酵母细胞能够在含有20μM COR的SD/-Ade/-His/-Leu/-Trp培养基上生长，说明在有JA-Ile的类似物COR存在的情况下，GhJAZ11-D与GhCOI1能发生相互作用(图3)。利用酵母双杂交和Pull-down实验验证GhJAZ11-D与GhMYC2蛋白的相互作用，结果显示GhJAZ11-D与GhMYC2结合后形成的二倍体酵母细胞能够在SD/-Ade/-His/-Leu/-Trp培养基上生长，说明在酵母系统中，GhJAZ11-D能与GhMYC2发生相互作用(图4A)；Pull-down实验证明，His-GhJAZ11-D蛋白与GST蛋白(对照)孵育纯化后没有检测到信号，相反，His-GhJAZ11-D蛋白与GST-GhMYC2蛋白孵育后，用His抗体进行检测，同Input一样，能够检测到结合蛋白信号(图4B)。以上结果说明，GhJAZ11-D蛋白与GhMYC2蛋白在体外能发生相互作用。
构建了CaMV 35S启动子驱动的GhJAZ11-D过量表达载体，转化棉花，获得过量表达JAZ11-D转基因棉花植株。RT-PCR分析表明，转基因棉花纤维中GhJAZ11-D表达水平都有不同程度的升高(图5A)。观察其植株的生长情况，发现转基因植株比野生型株高变高，节间距变长(图5B)。进一步观察其10天胚珠的大小，发现转基因棉花的胚珠相对野生型变小(图5C)。待棉花成熟，收取成熟纤维，测量分析发现纤维长度相对野生型变长，整齐度指数相对野生型升高，马克隆值相对野生型更接近于最佳值(图5D)。以上都说明提高GhJAZ11-D基因表达可以促进棉花纤维伸长，提高纤维品质。因此，推测GhJAZ11-D可能通过参与JA信号通路而调控棉花棉花纤维发育，进而影响棉纤维产量和品质。
本发明的优点：
1.提供了一个新的棉花JAZ11-D基因序列及cDNA序列。该基因在棉花纤维发育起始和伸长发育中都有较高水平的表达，表明该基因可能在控制棉纤维起始和伸长发育过程中起重要作用。
2.GhJAZ11-D蛋白定位于细胞核中，可以和JA信号通路受体GhCOI1和关键调控因子GhMYC2相互作用。
3.与野生型相比，过量表达GhJAZ11-D导致转基因棉花植株高度增加，胚珠变小，棉纤维长度增加，纤维品质有所改善，这表明GhJAZ11-D基因可能通过参与JA信号调控而在棉纤维细胞伸长发育过程中发挥重要作用，从而影响棉纤维产量和品质。
本发明通过以下附图和实施作进一步阐述，但不限制本发明的范围。
附图说明：
图1.荧光定量RT-PCR分析GhJAZ11-D在棉花组织中的表达。
R：根；H：下胚轴；S:茎；C：子叶；L：叶片；P：花瓣；A：花药；0O：开花当天胚珠。3O：开花后3天胚珠和纤维；6f：开花后6天纤维；9f：开花后9天纤维；12f：开花后12天纤维；15f：开花后15天纤维；18f：开花后18天纤维；20f：开花后20天纤维。
图2.GhJAZ11-D蛋白在棉花细胞中的亚细胞定位分析。
构建GhJAZ11-D与绿色荧光蛋白(eGFP)基因融合表达载体(GhJAZ11D:GFP)，转化棉花，获得转化愈伤组织，在共聚焦显微镜观察棉花愈伤组织细胞的GFP荧光，发现GFP荧光信号主要分布在细胞核中。A，显微镜明视野下的棉花愈伤组织细胞图像；B，愈伤组织细胞中GFP荧光信号；C，图A与图B的重合图像。
图3.GhJAZ11-D与GhCOI1蛋白相互作用验证
构建pGBKT7-GhJAZ11-D和pGADT7-GhCOI1酵母表达载体，分别转化酵母Y187和AH109菌株。利用酵母双杂交技术，将带有pGBKT7-GhJAZ11-D的Y187酵母细胞与带有pGADT7-GhCOI1的AH109酵母细胞接合生殖，分析子代二倍体在SD/-Leu/-Trp基本培养基上生长情况(左图)及SD/-Ade/-His/-Leu/-Trp/20μM COR选择培养基上生长情况(右图)。如右图的结果所示，GhJAZ11-D与GhCOI1蛋白能够发生相互作用，而GhJAZ11-D与pGBK7空载体(对照)不能发生相互作用。
图4.GhJAZ11-D与GhMYC2蛋白相互作用验证。
A，酵母双杂交技术验证GhJAZ11-D与GhMYC2蛋白相互作用。构建pGBKT7-GhJAZ11-D和pGADT7-GhMYC2酵母表达载体，分别转化酵母Y187和AH109菌株。将带有pGBKT7-GhJAZ11-D转化酵母Y187与带有pGADT7-GhMYC2的转化酵母AH109接合生殖，分析子代二倍体在SD/-Leu/-Trp培养基上生长情况(左图)及SD/-Ade/-His/-Leu/-Trp培养基上生长情况(右图)。如右图的结果所示，GhJAZ11-D与GhMYC2蛋白能够发生相互作用，而GhJAZ11-D与pGBK7空载体(对照)不能发生相互作用。本实验中pGADT1作为负对照。
B，pull-down实验分析验证GhJAZ11-D与GhMYC2的相互作用。His-GhJAZ11-D和GST-GhMYC2蛋白分别在大肠杆菌中表达纯化(详见实施方式)，然后进行pull-down分析。结果表明，GhJAZ11-D与GhMYC2蛋白能够发生相互作用(GST为负对照)。
图5.过量表达GhJAZ11-D转基因棉花表型分析。
A，荧光定量RT-PCR分析GhJAZ11-D基因在野生型和转基因棉花株系(L32和L44)5DPA(左图)和10DPA(右图)纤维中的表达(DPA表示开花后的天数)。B，野生型(WT)和GhJAZ11-D转基因棉花株系(L32和L44)成熟纤维长度比较。C，野生型(WT)和GhJAZ11-D转基因棉花株系(L32和L44)开花10天后种子(胚珠)的大小比较。D，野生型(WT)和GhJAZ11-D转基因棉花株系(L32和L44)成熟纤维品质检测分析。结果表明GhJAZ11-D在棉花纤维中的过量表达可以促进棉花纤维伸长，提高纤维品质。
具体实施方式：
一个棉花JAZ家族基因GhJAZ11-D全长序列的克隆鉴定及功能分析：
1.GhJAZ11-D基因及cDNA序列的分离鉴定
利用拟南芥JAZ家族基因和蛋白序列，通过比较分析，在中国农业科学院棉花研究所和南京农业大学公开的陆地棉基因组数据库中进行BLAST查询，鉴定获得30个棉花JAZ蛋白的基因，其中包括一条命名为GhJAZ11-D的基因序列。按照上述公共基因组数据库中的该基因序列设计引物，以棉花基因组DNA为模板，采用PCR技术扩增获得该基因的DNA全长序列，如SEQ ID NO.1所示，总长4559bp。根据该基因序列，设计引物，通过RT-PCR技术，以棉花纤维细胞总RNA为模板，分离获得GhJAZ11-D全长cDNA序列，如SEQ ID NO.2所示，总长1044bp。
2.定量RT-PCR分析GhJAZ11-D基因的表达
利用RNA提取试剂盒(Qiagin)提取纯化棉花不同组织和纤维的总RNA提取提取棉花纤维的总RNA。实时荧光定量RT-PCR研究基因的表达按照Li XB,Fan XP,Wang XL,Cai L,Yang WC,2005.The Cotton ACTIN1gene is functionally expressed in fibers andparticipates in fiber elongation.Plant Cell 17:859–875进行。首先，将棉花不同组织(根、下胚轴、茎、子叶、叶子、花瓣、花药、开花后0天胚珠、开花后3天纤维、6天纤维、9天纤维、12天纤维、15天纤维、18天纤维、20天纤维)的总RNA(2μg/样)用M-MLV ReverseTranscriptase(Promega)反转录成cDNA；然后，以cDNA为模板，用基因特异的引物(GhJAZ11-D RT-P1和GhJAZ11-D RT-P2)和Real-time PCR Master Mix(TOYOBO,Japan)进行定量PCR反应。以棉花GhUBI1基因作为RT-PCR反应的内标，计算目标基因表达水平的相对值。每一实验都重复3次，统计分析实验结果，见图1，结果表明GhJAZ11-D基因在棉花不同组织以及不同的纤维发育阶段都有一定的表达，而且在纤维起始期高量表达，在之后的纤维伸长期其表达量逐渐上升。
3.GhJAZ11-D蛋白的亚细胞定位分析
构建pCAMBIA2300-GhJAZ11-D-eGFP植物表达载体，将该载体通过电转化法转入LBA4404农杆菌中，再通过农杆菌介导的棉花遗传转化获得转化愈伤组织细胞。将培养3个月的棉花愈伤组织细胞置于激光共聚焦显微镜下观察GFP荧光，进行亚细胞定位分析，见图2，亚细胞定位分析表明，GhJAZ11-D蛋白定位于细胞核中。
4.酵母双杂交技术验证GhJAZ11-D与GhCOI1蛋白相互作用
构建pGBKT7-GhJAZ11-D载体和pGADT7-GhCOI1载体，分别转化酵母Y187和AH109细胞。将检测得到的阳性酵母菌落进行小规模的酵母结合实验，将二倍体子代酵母细胞涂布在SD/-Ade/-His/-Leu/-Trp/20μM COR选择培养基上。如果在上述选择培养基上能够生长，表明GhJAZ11-D与GhCOI1之间能够依赖于JA-Ile的类似物COR相互作用。否则，表明两者不能相互作用，结果见图4。如图4结果所示，GhJAZ11-D与GhCOI1蛋白能够发生相互作用，而GhJAZ11-D与pGBK7空载体(对照)不能发生相互作用。
5.酵母双杂交和pull-down技术验证GhJAZ11-D与GhMYC2蛋白相互作用
(1)酵母双杂交实验:构建pGBKT7-GhJAZ11-D载体和pGADT7-GhMYC2载体，分别转化酵母Y187和AH109细胞。将检测得到的阳性酵母菌落进行小规模的酵母结合实验，将二倍体子代酵母细胞涂布在SD/-Ade/-His/-Leu/-Trp选择培养基上。如果在选择培养基上能够生长，表明GhJAZ11-D与GhMYC2之间能够相互作用。否则，表明两者不能相互作用。结果显示GhJAZ11-D与GhMYC2结合后形成的二倍体酵母细胞能够在SD/-Ade/-His/-Leu/-Trp培养基上生长，说明在酵母系统中，GhJAZ11-D能与GhMYC2发生相互作用。
(2)pull-down实验:构建pET32a-GhJAZ11-D和pPGH-GhMYC2载体，分别转化大肠杆菌DE3细胞，诱导表达蛋白。将表达纯化的His-GhJAZ11-D蛋白及GST蛋白(对照)与还原型谷胱甘肽-琼脂糖柱子一起冰上振荡孵育1h；同样，His-GhJAZ11-D蛋白和GST-GhMYC2蛋白也与还原型谷胱甘肽-琼脂糖柱子在相同条件下孵育。孵育完成后，用GST蛋白纯化方法进行纯化，随后取20μL上清液进行SDS-PAGE电泳分析，His-GhJAZ11-D蛋白作为对照(Input)，完成后进行Western-blotting分析，将His抗体作为一抗，鼠抗作为二抗。如果出现与对照(Input)一样大小的条带(蛋白信号)，说明GhJAZ11-D与GhMYC2蛋白在体外能发生相互作用。否则，则说明两者不能相互作用。pull-down实验结果表明：GhJAZ11-D蛋白与GhMYC2蛋白在体外能发生相互作用。
6.GhJAZ11-D过量表达(OE)转基因棉花表型分析
构建CaMV 35S启动子驱动的pBI-GhJAZ11-D植物表达载体，转化农杆菌LBA4404。利用农杆菌浸染棉花下胚轴外植体，共培养2天后，将下胚轴外植体转移到选择培养基上培养，诱导并筛选转化愈伤组织。将该愈伤组织经过8–10个月的继代培养后，诱导分化出胚性愈伤组织和体细胞胚，体细胞胚进而萌发再生出转基因棉花小植株。将再生的棉花小植株移栽至土中，生长发育至开花结实。提取棉花基因组DNA，利用PCR技术检测鉴定阳性的转基因棉花植株。提取开花5天和10天后的棉花纤维RNA，利用定量RT-PCR技术分析转基因棉花中GhJAZ11-D基因表达水平。然后，选择不同表达水平的转基因棉花株系进行表型及遗传分析，结果见图5。结果表明，GhJAZ11-D在棉花纤维中的过量表达导致纤维长度显著增加，品质有所改善。
说明书核苷酸和氨基酸序列表
＜110＞ 华中师范大学
＜120＞ 棉花GhJAZ11-D基因鉴定及应用
＜160＞ 3
＜210＞ 1
＜211＞ 4559bp
＜212＞ DNA
＜213＞ 棉花 (Gossypium hirsutum)
＜400＞ 1
agtgtagatc taacctaaat aagcttgaat agccttttga agatcgggat acttctttcc 60
ggcacattcc ttgagcatcg actcgaaagc ccggctcaca aatccaccag ccgccatttc 120
gatctaagcc ggtgcttcga acacgataat tccggagttc ccttctctct caccctacta 180
aataaaaaaa taataatgcc gaaatggagg aattaagcaa tgatttttaa aaaagttgaa 240
gcagatcgtt gaagaagata gttaggatct acgagtgtat gatagcagaa ataatataaa 300
caacatacaa aaatgaatgt taccgcgctt tgttctaaga gggactaaat ttggagaaga 360
aattagaaaa agaaaagata ttgaagatct ggatacaaaa gagcagagca gatcagatca 420
gatgagatct gttgaaaatt gaattgatga tgatattctt ttacgaatat gagcgattac 480
tgggctttta gttgtgatga gcccagacga tgctcggttt tggcgtccaa aacagaacta 540
ataacgagca gatgatttgg agaatctttg gattaatttt tgttttattc aagatttgac 600
ctaaactttt cttacttttt cattttcatt aagctagatt ttgtttttaa catctttagt 660
attaaataag gttttgatta ttctcaactt aaatatttaa cttatttttt atattacacc 720
ttacaatata ctcgttatac tcacaatatt gttgcgaagt ttaaaaaaaa atcaattttt 780
tttatttttt cattttctct tgtatatatc tcttagttga ttgttgttaa actttttttt 840
atcattttca tttcaattat ttgagagaga gaaagagagc acttaagatt ttctagattt 900
tttagataac ttcttttctt tattgtttac ctcttttctt tgtattgtaa aagctatttg 960
agatatttta gatatcttca aaagcaaaat caacaaattt ttgtaggggt caaaattaaa 1020
ttgtaatttt tacgatagta aaaatatatt tttttaaatg attaaatcaa aattttatca 1080
tttttagagg aaccaatatg taattttatc tttattaatt taaaatttaa aaattttaaa 1140
tagacagaat ggaaaatttt tcatattagg ggaggcctgg gccttaccat cccccctaga 1200
tttgccccta tataggatct agttaagcca taaaaatttg gggaaggatt taccttagca 1260
tcatgaaaaa attacagatt gtaaatgtta tatattctcc ttgagaggta tcgattcaaa 1320
tataatcaat tgagaaatct ttagttgaga tatattaaag tgaatgtagg taattggggc 1380
agaataatta taaatcgaaa tgttcaagtt tttcatttat ttactcattc tttataaaat 1440
tttataaaga attttaaaat atcaattcac ccctcttgat attttcgagt ctaacaattg 1500
ttattctaat acaatatcaa atttttacat agtactaaca ttttagatta tcaaaacaaa 1560
cacaccatta gtttatcaaa ccatgtctaa aacacaaatc aataataaaa agattaaaac 1620
taataataag attgacacaa aatttaaaga cttaatattc taatagaaaa tattatacaa 1680
actataaaca gtcacccttg taattataac tcattgtgtg agtttttggc atgattatct 1740
aaatcgggcc aaatcgatcc attagattga gaattaatta aaatatcgat acggaaaaaa 1800
gctttagact aactgaattg tgaattagta caaatagatt gaattaacaa tggaataaat 1860
tgagataatt attaatattt ttatttttat aaatttataa taaattattt aattaaaccg 1920
ttaccttata aattcgacca tcaatccagg gtaaaaaaag agtccagggt cctgaatgat 1980
tgacacgttc caatcatcaa aacaaccacg acccttggat tggttcacgt gctttcactt 2040
tcacgtactt ttttattaat atattttatt cttttaaaat tccaaacaaa caatgtacct 2100
ttttttttta ttgatttcat ttccatatat ctttaaaacc aaattttggt ctgcctatag 2160
caagagtgaa aaaaatttta aaatttatca acgaaaagaa aggtcaaaag tctggtttcc 2220
atggaagaag aagctgaatc acgcgaggaa gtcaagccta acgtcgtcgt taaagagact 2280
aatggagata ttgtcggcga taacgatgca ggaaaattgg gtaccattga aacccctgat 2340
tttctttccc agaaaatatc ccataactgg taagctttaa ttgtattcat tccgtaaaaa 2400
aaaggagaca aatgtttctt tccggatctg ggtcttgata tatagctgtt taattatact 2460
cgatttgatg ttttaaaatg gaagtcttga tacatagtga aatttagtgc ttaattcatt 2520
accctaatct catctgtaga ggacaaattg aacatggttt taaaatgtac tactagtagt 2580
tttttttatt gatgttaaat ggtttgggct ggatttagtt agtgttaatc acttggaagc 2640
tttgaattcc cattgtataa aaaacggcga cctgatttct ttttatggta gtatttaaaa 2700
aaaacaaaaa aaaagggttt gtttggttat attttaggtt cgcatggtta cttactttag 2760
agggcttcca attctctctc tctctatatg tgtgtgtgtg tgtgtattcc cttttgatga 2820
aacctaggag atgttcatat gtcatattga aatttatact tatcggtgac tgactcagtt 2880
gagcatatgg tattcgattg tattgaagtt atagattcca taagaaaaat atatattagg 2940
aagtgcacat gcttgttcta tgttgtgttt cctcttcttt tcttttcttt tttttcattt 3000
aaaaccattt tgttatagcg aaaaattgta aagtattcaa gtgtcaccgt cttgacctga 3060
cccaacttgc agggcatcag gtgtgttgcg atagtataat cctttttggg cacccacagg 3120
tccctcaagg ttttgctacc ttcatatgtg actagccctc aaggtcccgc aggacagggc 3180
cgtatcatga tagcctaaac ctcctgttcg catatcattc tttgaagcaa aaattgttat 3240
ttttttctct attgcatcgc atgtagactt tacccctaaa gtaaattgtg cccagtagat 3300
ctttctcatc catgagtgaa gcaaaagggc acattagtca tatttctgca cacttctata 3360
atgttgtaag attttattct gctgttgtac tagttcccta ccgattctgg catctggagt 3420
aaataccaca agccttgctc aatcccaact caccatcttc tatggcggaa aagtttctgt 3480
atttgatgca atttccgctg aaaaggttgg cctttactta acattttaga atgttactat 3540
gttttggtag gatgagattt ttattccaaa gttaaccatc gatccatgtc tgtcagattc 3600
aagaaattat gcttattgct gctgctgtgg ctgctgctga tgttggctct gttgacatga 3660
agaatgctgc aaccgattat gccaccattt cacctgctct tacaaggtgt ccttcacttc 3720
aaagtactgc aactgctttg gcttcgccac aggcacagtt gtatcctttc cccaggacat 3780
cttttagcaa actgcaagct ggtaattcaa ataattacga ggttcaaatg caaaatattc 3840
tatgattttc atttttggtg catgtaagct aataatgtgg catcatgatt atgtgaatat 3900
caattggatc agaactacca attgcaagga gacactcact tcagcgtttt cttgagaagc 3960
ggcgggacag gtactcctcc gatcgtactc catcatttta ctgtttactt tataccccta 4020
agctatggca agtgtagtta tgtgtctgtt cccttttgta ggctggtgaa caagaaccca 4080
tatcctggtc catcaacacc aaaaatggtt gatggcacca aagctgacgt gagtgccaca 4140
acttcaccag agtctggctg cttcaaggca tcacccattc gtcaagaaga tatccaacca 4200
aaagctccag ctcatgttgc gtaaagctaa gcatatagga ttccttatat atatatattt 4260
gataaaaact gaatctagct ctgttattgt aagttacaaa gtgcagagga atacatctga 4320
tctgccactg gagttaggtg tggatagcaa gtgtacaatt aaacaggtct catggtagga 4380
tctgtccact tgtttcccta ttgccttctt gggcagcagt tttgatgttt cctaaacctg 4440
gacaccctct ctctgatctg cctgacaaat aacatttgcc atgtatgtat gattctctct 4500
gttctatggg tatgcaaaga ctgctaattt gtgtgtaacg tgttccaact ttgtacaat 4559
＜210＞ 1
＜211＞ 1044bp
＜212＞ DNA
＜213＞ 棉花 (Gossypium hirsutum)
＜400＞ 2
atggaagaag aagctgaatc acgcgaggaa gtcaagccta acgtcgtcgt taaagagact 60
aatggagata ttgtcggcga taacgatgca ggaaaattgg gtaccattga aacccctgat 120
tttctttccc agaaaatatc ccataactgt tccctaccga ttctggcatc tggagtaaat 180
accacaagcc ttgctcaatc ccaactcacc atcttctatg gcggaaaagt ttctgtattt 240
gatgcaattt ccgctgaaaa gattcaagaa attatgctta ttgctgctgc tgtggctgct 300
gctgatgttg gctctgttga catgaagaat gctgcaaccg attatgccac catttcacct 360
gctcttacaa ggtgtccttc acttcaaagt actgcaactg ctttggcttc gccacaggca 420
cagttgtatc ctttccccag gacatctttt agcaaactgc aagctgaact accaattgca 480
aggagacact cacttcagcg ttttcttgag aagcggcggg acaggctggt gaacaagaac 540
ccatatcctg gtccatcaac accaaaaatg gttgatggcg ccaaagctga cgtgagtgcc 600
acaacttcac cagagtctgg ctgcttcaag gcatcaccca ttcgtcaaga agatatccaa 660
ccaaaagctc cagctcatgt tgcgtaaagc taagcatata ggattcctta tatatatata 720
tttgataaaa actgaatcta gctctgttat tgtaagttac aaagtgcaga ggaatacatc 780
tgatctgcca ctggagttag gtgtggatag caagtgtaca attaaacagg tctcatggta 840
ggatctgtcc acttgtttcc ctattgcctt cttgggcagc agttttgatg tttcctaaac 900
ctggacaccc tctctctgat ctgcctgaca aataacattt gccatgtatg tatgattctc 960
tctgttctat gggtatgcaa agactgctaa tttgtgtgta acgtgttcca actttgtaca 1020
ataaagaaaa aaaaaaaaaa aaaa 1044
＜210＞ 1
＜211＞ 228
＜212＞ PRT
＜213＞ 棉花 (Gossypium hirsutum)
＜400＞ 3
Met Glu Glu Glu Ala Glu Ser Arg Glu Glu Val Lys Pro Asn Val
1 5 10 15
Val Val Lys Glu Thr Asn Gly Asp Ile Val Gly Asp Asn Asp Ala
20 25 30
Gly Lys Leu Gly Thr Ile Glu Thr Pro Asp Phe Leu Ser Gln Lys
35 40 45
Ile Ser His Asn Cys Ser Leu Pro Ile Leu Ala Ser Gly Val Asn
50 55 60
Thr Thr Ser Leu Ala Gln Ser Gln Leu Thr Ile Phe Tyr Gly Gly
65 70 75
Lys Val Ser Val Phe Asp Ala Ile Ser Ala Glu Lys Ile Gln Glu
80 85 90
Ile Met Leu Ile Ala Ala Ala Val Ala Ala Ala Asp Val Gly Ser
95 100 105
Val Asp Met Lys Asn Ala Ala Thr Asp Tyr Ala Thr Ile Ser Pro
110 115 120
Ala Leu Thr Arg Cys Pro Ser Leu Gln Ser Thr Ala Thr Ala Leu
125 130 135
Ala Ser Pro Gln Ala Gln Leu Tyr Pro Phe Pro Arg Thr Ser Phe
140 145 150
Ser Lys Leu Gln Ala Glu Leu Pro Ile Ala Arg Arg His Ser Leu
155 160 165
Gln Arg Phe Leu Glu Lys Arg Arg Asp Arg Leu Val Asn Lys Asn
170 175 180
Pro Tyr Pro Gly Pro Ser Thr Pro Lys Met Val Asp Gly Ala Lys
185 190 195
Ala Asp Val Ser Ala Thr Thr Ser Pro Glu Ser Gly Cys Phe Lys
200 205 210
Ala Ser Pro Ile Arg Gln Glu Asp Ile Gln Pro Lys Ala Pro Ala
215 220 225
His Val Ala
228
Claims (3)
1. 棉花GhJAZ11-D基因，其特征在于，序列如SEQ.ID.No.1所示，基因的基因组序列包括5’-上游启动子区、4个内含子区和5个外显子区以及3’-非翻译区，5’-上游启动子区为第1 – 2220bp，第一内含子区为第2370 – 3393bp，第二内含子区为第3506 – 3596bp，第三内含子区为第3802 – 3912bp，第四内含子区为第3971 – 4061bp，3’-非翻译区(UTR) 为第4225 – 4559bp，基因编码区为第2221-4224bp。
2. 权利要求1所述的棉花GhJAZ11-D基因的cDNA，其特征在于：序列如SEQ.ID.No.2所示，基因编码区 (ORF)从第1–687bp，起始密码子为第1-3bp，终止密码子为第685-687bp。
3.权利要求1所述的棉花GhJAZ11-D基因编码的蛋白质，其特征在于：所述蛋白质的编码基因为四倍体棉花基因，能够和JA信号通路受体GhCOI1和关键调控因子GhMYC2相互作用，序列如SEQ.ID.No.3所示。
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| CN104711266A (zh) * | 2015-03-31 | 2015-06-17 | 华中师范大学 | 一个棉花bHLH转录因子基因GhFP1及其启动子的鉴定及应用 |
| CN105906696A (zh) * | 2016-06-03 | 2016-08-31 | 华中师范大学 | 一个新的棉纤维发育相关基因GhEIN3的鉴定及应用 |
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| CN104152464A (zh) * | 2014-08-01 | 2014-11-19 | 华中师范大学 | 一个棉纤维特异性mybl1基因 |
| CN104711266A (zh) * | 2015-03-31 | 2015-06-17 | 华中师范大学 | 一个棉花bHLH转录因子基因GhFP1及其启动子的鉴定及应用 |
| CN105906696A (zh) * | 2016-06-03 | 2016-08-31 | 华中师范大学 | 一个新的棉纤维发育相关基因GhEIN3的鉴定及应用 |
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