CN111676235B - GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用 - Google Patents
GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用 Download PDFInfo
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- CN111676235B CN111676235B CN202010562774.9A CN202010562774A CN111676235B CN 111676235 B CN111676235 B CN 111676235B CN 202010562774 A CN202010562774 A CN 202010562774A CN 111676235 B CN111676235 B CN 111676235B
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Abstract
本发明属于植物基因工程技术领域，具体涉及GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用。本发明要解决的技术问题是为改良棉花的品质提供一种新选择。本发明的技术方案是GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用。利用本发明的基因对棉花进行改良，能够实现棉花纤维长度的明显提高。
Description
技术领域
本发明涉及植物基因工程技术领域，具体涉及GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用。
背景技术
棉花是世界上重要的经济作物。在中国乃至世界经济发展中都具有十分重要的地位。棉花纤维是纺织中主要的天然纤维原料，其纤维制品具有可降解、吸湿、透气和保暖等诸多优良特性。我国是世界上的棉花生产大国，常年种植面积超过300万公顷，年产量在600万吨左右。尽管如此，我国对棉花需求量巨大，常年存在约25％的需求缺口通过从国外进口弥补。我国也因此是全球最大的棉花进口国(卢秀茹et al.,2018)。另一方面，为适应技术设备的更新以及人们对棉制品品质需求的提高，高品质的纤维(“长、强、细”)一直是我国纺织工业所需以及育种工作的重点(袁有禄et al.,2018)。因此，提高棉花产量和改善纤维品质，这对于我国棉花产业和棉纺织业可持续发展具有十分重要的意义。
运用现代基因工程育种，在定向改良和加快作物育种进程上相较传统育种具有明显的优势。通过表达来源于苏云金芽孢杆菌的Bt毒蛋白编码基因的抗虫棉的培育成功也证实了这一新兴手段的可行性。要对棉花纤维性状进行基因工程改良，目的基因的选取是其关键。2009年Wang等利用在棉花中下调肌动蛋白解聚因子(GhADF1)的表达，成功提高了转基因棉花纤维长度和强度(Wang et al.,2009)。向棉花中导入醋杆菌的纤维素合成的基因acsA和acsB，纤维长度和强度提高约15％，而纤维细度降低了约12％(Li et al.,2004)。2011年Zhang等人通过在纤维起始阶段的胚珠表皮特异表达来源于农杆菌的生长素合成酶基因iaaM，实现了纤维产量和马克隆值的同步改良(Zhang et al.,2011)。Jiang等(2012年)利用蔗糖合酶基因GhSusA1在棉花中超量表达，提高了棉纤维的长度和强度，同时纤维的产量也有所提高(Jiang et al.,2011)。另一个研究组也利用土豆的蔗糖合酶基因在棉花中表达，也表明该类蛋白在纤维长度上的改良作用(Xu et al.,2012)。对纤维扩张因子GhEXPA8的研究表明，超量表达该基因对纤维长度和马克隆值有明显改良(Bajwa et al.,2015)。到目前为止，这些可供用于棉花纤维性状基因工程改良的基因仍然很少，部分结果还尚待田间实验的评估。大量的发现于改良纤维品质有价值的基因十分必要。
小GTP结合蛋白ROP是一类植物特有的GTP酶，控制着植物器官的形态建成。ROP蛋白主要通过在GTP结合形态(激活态)和GDP结合态之间转换，作为分子开关发挥调控作用(Kost,2008)。人为对ROP蛋白15位甘氨酸(G15)或64位的谷氨酰胺(Q64)进行定点突变能够让该蛋白组成性激活，表现出近似超量表达ROP的表型；而对ROP蛋白20位的苏氨酸(T20)或121位的天冬氨酸(D121)进行点突变则能够让该蛋白显性失活，表现出近似基因表达受到抑制的表型(Feiguelman et al.,2018)。
发明内容
本发明要解决的技术问题是为改良棉花的纤维提供一种新选择。
本发明的技术方案是GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用。
本发明还提供了GTP结合蛋白基因GhROP6在改良棉花的纤维长度中的应用。
具体的，所述的GTP结合蛋白具有如SEQ ID No.3或SEQ ID No.4所示的氨基酸序列。
具体的，表达小GTP结合蛋白基因GhROP6具有如SEQ ID No.1或SEQ ID No.2所示的核苷酸序列。
本发明还提供了改良棉花纤维长度的方法，包括如下步骤：
a、构建载体：构建表达小GTP结合蛋白基因GhROP6和GhROP6组成性激活突变基因的载体；
b、遗传转化：将步骤a得到的载体转化棉花，即得到纤维长度增长的棉花。
具体的，所述的表达小GTP结合蛋白具有如SEQ ID No.3所示的氨基酸序列，GhROP6组成性激活突变蛋白具有SEQ ID No.4所示的氨基酸序列。
具体的，所述小GTP结合蛋白基因GhROP6具有如SEQ ID No.1所示的核苷酸序列；所述GhROP6组成性激活突变基因具有如SEQ ID No.2所示的核苷酸序列。
具体的，所述的小GTP结合蛋白基因或其组成性结合突变体基因在组成型启动子CaMV35S的介导下表达。
本发明还提供了改良棉花纤维长度的组合物，其主要活性成分为表达小GTP结合蛋白基因GhROP6和GhROP6组成性激活突变基因的载体；和/或含有所述载体的宿主细胞。
具体的，所述的表达小GTP结合蛋白具有如SEQ ID No.3所示的氨基酸序列，GhROP6组成性激活突变蛋白具有SEQ ID No.4所示的氨基酸序列。
具体的，所述小GTP结合蛋白基因GhROP6具有如SEQ ID No.1所示的核苷酸序列；所述GhROP6组成性激活突变基因具有如SEQ ID No.2所示的核苷酸序列。
本发明所涉及的蛋白GhROP6是ROP家族的一个蛋白，和拟南芥的AtROP6同源。过去研究表明，激活的ROP6能够抑制生长素运输蛋白PIN2的内吞到胞内，从而让PIN2更多的保留在质膜上(Lin et al.,2012)，从而导致生长素不能及时运输到细胞内。但在本发明中发现棉花来源的GhROP6却有相反的效果。GhROP6的超量表达或者激活能够促进GhPIN3a蛋白的降解(图4和图5)。GhPIN3a和PIN2一样，是一个质膜定位蛋白控制生长素从胞内流向胞外的运输蛋白，控制着棉花胚珠表皮以纤维为中心的生长素浓度的建立。纤维细胞中GhPIN3a蛋白的定位缺失，让生长素不能流出胞外而在纤维细胞中聚集，从而促进纤维细胞的发育(Zeng et al.,2019)。而在GhROP6超量表达或者激活的棉花中，GhPIN3a的质膜定位受到进一步削弱，从而使棉花纤维的发育得到促进，成熟纤维长度增加。
本发明的有益效果：
利用本发明载体对棉花进行改良，纤维品质性状表现为纤维长度增加，纤维品质得到明显改良。这将为纺织工业带来更好的棉花纤维原料，可带来巨大的经济效益。
附图说明
图1：GhROP6植物表达载体构建示意图；
植物表达载体pLGN，是以常用的pCambia2300植物表达为骨架改造的一个双元载体(具体改造过程见参考文献Zeng et al.,2019)；其T-DNA区段边界一端(T-Border)附近有加倍的组成型启动子35S(2×35S)控制的标记基因NPT II和报告基因GUS的融合基因，以及Nos终止子序列；其T-DNA区域另一端(T-Border)附近有组成型启动子35S和polyA(具有转录终止的作用)；为转基因植株鉴定方便，在35S和polyA中添加有一个荧光蛋白基因mCherry。
图2：CA-ghrop6植物表达载体构建流程图。
图3：转基因棉花开花当天胚珠中mCherry基因的表达量；
Wild type：野生型棉花，作为无信号的阴性对照；OX：超量表达GhROP6的转基因棉花；CA：超量表达CA-ghrop6的转基因棉花。
图4：转基因棉花胚珠表皮细胞GhPIN3a::eYFP质膜定位情况；
GhPIN3a::eYFP：与35S::GhROP6转基因棉花杂交后代分离出的仅表达GhPIN3a::eYFP的转基因棉花；GhPIN3a::eYFP GhROP6：同时表达35::GhPIN3a::eYFP和35S::GhROP6转基因棉花胚珠；GhPIN3a::eYFP CA-ghrop6:同时表达35::GhPIN3a::eYFP和35S::CA-ghrop6转基因棉花胚珠；标尺为20μm。
图5：转基因棉花胚珠表皮细胞GhPIN3a::eYFP质膜定位信号强度；
GhPIN3a::eYFP：与35S::GhROP6转基因棉花杂交后代分离出的仅表达GhPIN3a::eYFP的转基因棉花；GhPIN3a::eYFP GhROP6：同时表达35::GhPIN3a::eYFP和35S::GhROP6转基因棉花胚珠；GhPIN3a::eYFP CA-ghrop6:同时表达35::GhPIN3a::eYFP和35S::CA-ghrop6转基因棉花胚珠；*：和单独表达GhPIN3a:eYFP的样品进行学生t测验比较，显著性P<0.05。图6：转基因棉花成熟纤维长度；
Wild type：野生型棉花，作为无信号的阴性对照；OX：超量表达GhROP6的转基因棉花；CA：超量表达CA-ghrop6的转基因棉花；标尺指示1cm。
图7：转基因棉花成熟纤维长度统计；
Wild type：野生型棉花，作为无信号的阴性对照；OX：超量表达GhROP6的转基因棉花；CA：超量表达CA-ghrop6的转基因棉花；*：和野生型进行学生t测验比较，显著性P<0.05。
具体实施方式
以下结合附图对本发明进行进一步的详细说明，但以下说明并不对本发明进行限定，任何对本发明的变形和改变，只要不脱离本发明的精神，均应属于本发明所附权利要求所定义的范围。
本发明实例中的试剂药品未做具体说明的均为普通市售。材料方法未做具体说明的均参考《分子克隆实验指南》(Sambrook和Russell,2001)。
实施例1载体的构建
1.1植物RNA提取
选取约1g新鲜的棉花材料在研钵中加入液氮充分碾磨成细粉，用艾德莱生物公司的EASYspin Plant RNAKit提取RNA，提取步骤严格按照内附说明书进行。提取完成后取2μL进行琼脂糖凝胶电泳，以检测所提取RNA的质量。
1.2 cDNA合成
RNA提取完成后，用TaKaRa公司的PrimeScript RT reagent Kit with gDNAEraser进行cDNA合成，操作步骤按照说明书进行。
1.3棉花基因GhROP6的获得
根据GhROP6的核苷酸序列(SEQ ID NO.1，JGI登录号：Gohir.A01G168200)，设计引物SEQ ID NO.6和SEQ ID NO.7，从陆地棉开花当天的胚珠cDNA中直接扩增获得GhROP6的基因。回收GhROP6片段后，将该片段连接到pTOPO-Blunt克隆载体，测序验证后用于后续载体构建使用。
SEQ ID NO.6，GhROP6扩增上游引物1：5’-atgagtgcatcaaggttcatca-3’；
SEQ ID NO.7，GhROP6扩增下游引物2：5’-tcacaatatcgagcaggcctttt-3’。
SEQ ID NO.1：基因GhROP6(JGI登录号：Gohir.A01G168200)，来源于陆地棉；翻译后蛋白质的序列如SEQ ID NO.3所示。
1.4 GhROP6植物超量表达载体的构建
构建流程见图1，用于构建所述植物表达载体的骨架载体包括pLGN载体(pLGN载体改造过程见参考文献Zeng et al.,2019)。本发明所述植物表达载体的结构优选如图1所示，NPTII：新霉素磷酸转移酶基因；GUS：β-葡萄糖酸苷酶基因；35S：来源于花椰菜花叶病毒的植物组成性启动子；nos Terminator：冠瘿碱合成酶基因终止子；35S Terminator：35S基因终止子；LB：T-DNA左边界；RB：T-DNA右边界；用于构建植物表达载体的骨架载体为pLGN载体，具有CaMV 35S启动子调控下的GUS基因，便于在植物遗传转化的过程中对转化子进行GUS染色的筛选。
将由pCambia2300载体改造而来的pLGN植物表达载体用BamHⅠ和KpnⅠ进行酶切线性化。各种限制性内切酶为Thermo Scientific公司产品，按照说明书进行酶切。设计带同源臂的引物扩增mCherry序列，分别为SEQ ID NO.8(5’-acagggtacggatccacaattaccaacaacaacaa-3’)和SEQ ID NO.9(5’-tctagacccggggaattcggag-3’)。同时设计带同源臂的引物扩增GhROP6序列，引物分别为SEQ ID NO.10(5’-ttccccgggtctagaatgagtgcatcaaggttcat-3’)和SEQ ID NO.11(5’-catgtcgacggatcctcacaatatcgagcaggcct-3’)。将pLGN线性化载体、mCherry和GhROP6 PCR扩增产物按分子数比1:3:3混合，在同源重组酶的催化下，37℃反应30min后进行大肠杆菌转化，由此完成pLGN::35S::GhROP6植物表达载体的构建。将GhROP6构建在pLGN植物表达载体中用于棉花遗传转化，所用的棉花实验材料为柯字棉，来自陆地棉(Gossypium hirsutum L)。
1.5 35S::CA-ghrop6植物表达载体的构建：
根据ROP蛋白序列特征和文献报道，采用第15位甘氨酸(Gly)突变成缬氨酸(Val)，构建成GhROP6的组成性激活突变体(Constitutively Active，CA)，GhROP6激活态基因GhROP6G15V的核苷酸序列如SEQ ID NO.2所示，GhROP6激活态基因GhROP6G15V翻译后的蛋白氨基酸序列如SEQ ID No.4所示。
具体通过PCR的形式将GhROP6的44位核苷酸由鸟嘌呤(g)突变为胸腺嘧啶(t)。设计的突变引物SEQ ID NO.12(5’-cactgttggtgacgttgccgtcggcaagac-3’)和SEQ ID NO.13(5’-gtcttgccgacggcaacgtcaccaacagtg-3’)，以pLGN::35S::GhROP6载体质粒为模板进行扩增，形成带有相应突变位点的PCR产物。扩增产物用DpnⅠ在37℃条件下处理30min，将质粒模板消化掉，从而使有突变位点的PCR产物得到富集。将消化产物加入到同源重组体系中，使线性的PCR产物通过同源重组形成环状，37℃反应30min后即可进行大肠杆菌转化，由此完成35S::CA-ghrop6植物表达载体的构建。
实施例2转化棉花
2.1用电激法将构建的植物表达载体质粒导入农杆菌GV3101
参考Bio-RAD MicroPulser用户说明书，将上述载体通过电激转化法导入农杆菌GV3101。
农杆菌电击转化步骤为：取出农杆菌感受态细胞置于冰上融化，电击转化所用的电击杯用无菌水清洗5～6次，置于冰上备用。吸取20～50ng质粒与感受态细胞混匀后加入电击杯中。打开Bio-Rad公司的电击转化仪，选择Agr/Bacteria模式进行电击。电击后立即加入800μL YEB液体培养基，28℃，200rpm振荡培养3h。10000rpm离心1min，弃上清，留下约100μL培养基重悬菌体，菌液均匀涂布在加有相应抗生素(如50μg/mL Kan)的YEB平板上，28℃恒温倒置培养48h。
2.2组成型表达GTP结合蛋白GhROP6和激活突变体CA-ghrop6载体整合到棉花基因组
通过根癌农杆菌介导的方法进行棉花的遗传转化(转化过程中所用的培养基见表1)，上述植物表达载体的T-DNA区段通过农杆菌介导胚性愈伤的方法导入棉花。具体方法如下：
(1)棉花种子萌发：陆地棉栽培种柯字棉种子剥去外壳，选取大而饱满的子仁置于三角瓶中，先用75％酒精灭菌1～2min，用无菌水漂洗2次；再用0.1％升汞(HgCl2)灭菌10min，无菌水漂洗6次。用无菌水于常温，120rpm摇床上振荡培养12h。将露白的子仁胚根朝下接种入萌发培养基中，并置于28℃黑暗条件下萌发2～3d，以获得无菌棉花幼苗。
(2)转化农杆菌的培养:将含有上述植物表达载体的农杆菌菌种，接种于含50mg/L卡那霉素和125mg/L链霉素的YEB固体培养基(0.5％蔗糖(W/V)，0.1％细菌用酵母提取物(W/V)，1％细菌用胰化蛋白胨(W/V)，0.05％MgSO4·7H2O(W/V)，1.5％的琼脂粉(W/V)pH7.0)上划线培养。接种新划板活化的农杆菌单菌落于5mL含相同抗生素的YEB液体培养基中，28℃、，200rpm振荡培养过夜。取1mL菌液于20～25mL含相同抗生素的YEB液体培养基中进行二次活化，28℃，200rpm振荡培养至OD600为0.8～1.0左右。10000rpm室温离心1min，收集菌体，用等体积的液体共培养基(含100mg/L乙酰丁香酮AS)重悬菌体，作为侵染液备用。
(3)浸染和共培养:将棉花无菌下胚轴切成0.5cm左右的小段，置于含侵染液的三角瓶内，28℃、100rpm摇床侵染45min。弃去菌液，将侵染过的下胚段接种入固体共培养基上(含100mg/L乙酰丁香酮，AS)，28℃黑暗培养2d。
(4)转化子的筛选:共培养完成后将下胚段转移到筛选培养基上进行脱菌和选择培养，28℃，16h光照下培养,每2周继代一次。1～2个月后大部分愈伤组织褐化死亡，少部分表现出卡那霉素抗性，生长出新鲜的胚性愈伤组织。将愈伤组织块进行继代，每块组织增殖到2.0～3.0g时接入液体悬浮培养基中，在摇床上120rpm振荡悬浮培养以获得大量体胚。悬浮2周后用30目筛网过滤悬浮培养组织，网下沉淀物转接入体胚成熟培养基。萌发的成熟胚转入体胚伸长诱导培养基上，于28℃黑暗条件下培养2周以诱导体胚伸长、萌发。取较大的萌发胚(>0.5cm)转接入SH培养基成苗,28℃，16h光照下培养。待幼苗长到约2cm高时，剪取幼苗嫁接到有3～4片真叶的棉花幼苗上。
表1根癌农杆菌介导的棉花遗传转化用培养基
Gelrite：Sigma，货号：G1910；SH：Schenk&Hildebrandt，1972。
2.3获得纤维性状改良的棉花
获得T1代的转基因棉花在温室中培养，常规管理。收集成熟后的纤维进行性状分析。获得的转基因棉花在表型和生长发育上与野生型的对照没有明显区别。
实施例3 GhROP6和CA-ghrop6超量表达转基因棉花目的基因表达量筛选
为了鉴定方便，mCherry基因和GhROP6或CA-ghrop6在表达载体中共用一套启动子和终止子。因此，可以用mCherry基因的表达量评价转基因引入的GhROP6或CA-ghrop6的表达情况。用Real-time PCR的方法对35S::GhROP6和35S::CA-ghrop6转基因植株中mCherry基因的表达量进行筛选。分别提取野生型棉花、GhROP6和CA-ghrop6超量表达转基因棉花开花当天胚珠的总RNA，通过逆转录合成cDNA单链。以此为模板进行real-time PCR扩增，对35S::GhROP6和35S::CA-ghrop6转基因植株中mCherry基因进行表达量筛选鉴定。
具体操作步骤为：
①cDNA模板的质量检测：将合成的cDNA加蒸馏水稀释到50μL，取2μL的cDNA作为模板，先进行棉花内参基因GhHIS3扩增，GhHIS3的引物为SEQ ID NO.14(5’-gaagcctcatcgataccgtc-3’)和SEQ ID NO.15(5’-ctaccactaccatcatggc-3’)。用实时定量PCR仪进行扩增，检测cDNA模板的浓度，将所有模板扩张结果Ct值调整在16～20之间，以满足后续表达量分析。
②目的基因的表达量检测：以棉花GhHIS3作为内参基因，检测目的基因mCherry的表达量，mCherry的引物为SEQ ID NO.16(5’-caggacggcgagttcatcta-3’)和SEQ ID NO.17(5’-gtcttgacctcagcgtcgta-3’)。每个样品重复3次进行扩增，同时以蒸馏水为模板作为阴性对照。
③扩增体系(20μL)：2×ChamQ SYBR qPCR Master Mix 10μL，内参基因和目的基因上下游特异性引物各0.5μL，cDNA模板2～9μL，加蒸馏水至20μL。
④扩增程序：95℃预变性3min；95℃变性30sec，60℃退火延伸30sec，共40个循环；最后以溶解曲线检测引物的特异性。
结果显示(图3)，在35S::GhROP6和35S::CA-ghrop6转基因棉花胚珠中均能检测到mCherry的明显表达，远高于而作为阴性对照的野生型样本扩增出的背景信号，表明目的基因GhROP6和CA-ghrop6基因在转基因棉花中超量表达。选取表达量相对较高的35S::GhROP6转基因棉花的14和33号转化子和35S::CA-ghrop6转基因棉花的21和22号转化子进行后续的表型鉴定。
实施例5转基因棉花对棉花细胞中GhPIN3a质膜定位强度的分析
取GhPIN3a::eYFP分别与GhROP6和CA-ghrop6杂交的转基因棉花开花当天的胚珠，置于激光共聚焦显微镜下观察胚珠表皮细胞质膜上GhPIN3a::eYFP的荧光信号(图4)。激发光为514nm，发射光为520-560nm，物镜为40×油镜。通过Image J软件统计GhPIN3a::eYFP在质膜的荧光强度，每个胚珠随机选取30个细胞，每个转基因棉花随机选取9个胚珠进行统计(图5)。
实施例6转基因棉花成熟纤维长度测定
取35S::GhROP6、35S::CA-ghrop6转基因和野生型棉花的成熟棉籽各10颗，用梳子将棉籽上纤维梳理后，测量纤维长度。
结果显示(图6和7)，野生型棉花成熟纤维长度为29.3mm，而35S::GhROP6转基因棉花纤维长度为31.3mm(OX14)和31.4mm(OX33)。OX33转基因棉花纤维长度较野生型增长2.1mm。35S::CA-ghrop6转基因棉花纤维长度为30.0(CA21)和31.9mm(CA22)。CA22转基因棉花纤维长度比野生型增长2.6mm。这表明35S::GhROP6和35S::CA-ghrop6转基因棉花成熟纤维长度得到明显改良。
综上所述，表达GTP结合蛋白基因GhROP6和激活突变体CA-ghrop6能够促进纤维细胞起始更快，同时使棉花纤维长度得到明显改良。
上述实施例表明，本发明通过对GTP结合蛋白基因GhROP6和激活突变体CA-ghrop6在棉花中的组成性表达，能够促进纤维细胞起始更快，提高成熟纤维长度，纤维品质得到明显改良。本发明方法简便易行，效果显著，具有很好的市场前景。
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SEQUENCE LISTING
<110> 西南大学
<120> GTP结合蛋白基因GhROP6在调控棉花纤维性状中的应用
<130> 2020
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 597
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<220>
<223> 基因GhROP6
<400> 1
atgagtgcat caaggttcat caaatgtgtc actgttggtg acggtgccgt cggcaagact 60
tgcatgctca tctcctacac cagcaatact ttccctactg attatgtgcc aactgtcttt 120
gacaacttta gcgcaaatgt ggttgtggac ggcaacactg ttaatcttgg attgtgggat 180
actgctggcc aggaagacta caatagatta agacctttga gctatcgtgg agcagatgtc 240
ttcttgttgg cattctctct cattagcaaa gccagctacg aaaacgtttc taagaaatgg 300
attccggagt tgaggcatta tgcacctggt gttccaatta ttcttgttgg gactaagctt 360
gatcttcggg atgataagca gttcttcgta gatcaccctg gagcagtgcc cattaccaca 420
gcccaggggg aggaattaag aaagctgatt ggagctcctg tttacattga atgtagttca 480
aagacacagc agaacgtgaa agcagtcttc gatgcggcca tcaaagtggt tctccagcca 540
ccaaagcaga agaagaaaaa gaagagaaag tcacaaaagg cctgctcgat attgtga 597
<210> 2
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<212> DNA
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<220>
<223> GhROP6激活态基因
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atgagtgcat caaggttcat caaatgtgtc actgttggtg acgttgccgt cggcaagact 60
tgcatgctca tctcctacac cagcaatact ttccctactg attatgtgcc aactgtcttt 120
gacaacttta gcgcaaatgt ggttgtggac ggcaacactg ttaatcttgg attgtgggat 180
actgctggcc aggaagacta caatagatta agacctttga gctatcgtgg agcagatgtc 240
ttcttgttgg cattctctct cattagcaaa gccagctacg aaaacgtttc taagaaatgg 300
attccggagt tgaggcatta tgcacctggt gttccaatta ttcttgttgg gactaagctt 360
gatcttcggg atgataagca gttcttcgta gatcaccctg gagcagtgcc cattaccaca 420
gcccaggggg aggaattaag aaagctgatt ggagctcctg tttacattga atgtagttca 480
aagacacagc agaacgtgaa agcagtcttc gatgcggcca tcaaagtggt tctccagcca 540
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<210> 3
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Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala
1 5 10 15
Val Gly Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro
20 25 30
Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val
35 40 45
Val Asp Gly Asn Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln
50 55 60
Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val
65 70 75 80
Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val
85 90 95
Ser Lys Lys Trp Ile Pro Glu Leu Arg His Tyr Ala Pro Gly Val Pro
100 105 110
Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe
115 120 125
Phe Val Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala Gln Gly Glu
130 135 140
Glu Leu Arg Lys Leu Ile Gly Ala Pro Val Tyr Ile Glu Cys Ser Ser
145 150 155 160
Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile Lys Val
165 170 175
Val Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Lys Arg Lys Ser Gln
180 185 190
Lys Ala Cys Ser Ile Leu
195
<210> 4
<211> 198
<212> PRT
<213> artificial
<220>
<223> GhROP6激活态基因GhROP6G15V翻译后的蛋白氨基酸序列
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Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Val Ala
1 5 10 15
Val Gly Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro
20 25 30
Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val
35 40 45
Val Asp Gly Asn Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln
50 55 60
Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val
65 70 75 80
Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val
85 90 95
Ser Lys Lys Trp Ile Pro Glu Leu Arg His Tyr Ala Pro Gly Val Pro
100 105 110
Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe
115 120 125
Phe Val Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala Gln Gly Glu
130 135 140
Glu Leu Arg Lys Leu Ile Gly Ala Pro Val Tyr Ile Glu Cys Ser Ser
145 150 155 160
Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile Lys Val
165 170 175
Val Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Lys Arg Lys Ser Gln
180 185 190
Lys Ala Cys Ser Ile Leu
195
<210> 5
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<213> artificial
<220>
<223> 35S启动子
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ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt gctttgaaga 120
cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg 180
accactgtcg gcagaggcat cttcaacgat ggcctttcct ttatcgcaat gatggcattt 240
gtaggagcca ccttcctttt ccactatctt cacaataaag tgacagatag ctgggcaatg 300
gaatccgagg aggtttccgg atattaccct ttgttgaaaa gtctcaattg ccctttggtc 360
ttctgagact gtatctttga tatttttgga gtagacaagc gtgtcgtgct ccaccatgtt 420
gacgaagatt ttcttcttgt cattgagtcg taagagactc tgtatgaact gttcgccagt 480
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<210> 6
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<220>
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<220>
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<220>
<223> 扩增mCherry的引物序列2
<400> 9
tctagacccg gggaattcgg ag 22
<210> 10
<211> 35
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<213> artificial
<220>
<223> 扩增带同源臂的GhROP6序列的引物1
<400> 10
ttccccgggt ctagaatgag tgcatcaagg ttcat 35
<210> 11
<211> 35
<212> DNA
<213> artificial
<220>
<223> 扩增带同源臂的GhROP6序列的引物2
<400> 11
catgtcgacg gatcctcaca atatcgagca ggcct 35
<210> 12
<211> 30
<212> DNA
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<220>
<223> 突变引物1
<400> 12
cactgttggt gacgttgccg tcggcaagac 30
<210> 13
<211> 30
<212> DNA
<213> artificial
<220>
<223> 突变引物2
<400> 13
gtcttgccga cggcaacgtc accaacagtg 30
<210> 14
<211> 20
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<220>
<223> 扩增GhHIS3的引物1
<400> 14
gaagcctcat cgataccgtc 20
<210> 15
<211> 19
<212> DNA
<213> artificial
<220>
<223> 扩增GhHIS3的引物2
<400> 15
ctaccactac catcatggc 19
<210> 16
<211> 20
<212> DNA
<213> artificial
<220>
<223> 检测mCherry的引物1
<400> 16
caggacggcg agttcatcta 20
<210> 17
<211> 20
<212> DNA
<213> artificial
<220>
<223> 检测mCherry的引物2
<400> 17
gtcttgacct cagcgtcgta 20
Claims (5)
1.过表达GTP结合蛋白基因GhROP6在增长棉花纤维长度中的应用，其特征在于，所述GTP结合蛋白的氨基酸序列如SEQ ID No.3或SEQ ID No.4所示。
2.如权利要求1所述的应用，其特征在于：表达GTP结合蛋白基因GhROP6的核苷酸序列如SEQ ID No.1或SEQ ID No.2所示。
3.改良棉花纤维长度的方法，其特征在于：包括如下步骤：
a、构建载体：构建表达小GTP结合蛋白基因GhROP6和GhROP6组成性激活突变基因的载体；所述表达小GTP结合蛋白的氨基酸序列如SEQ ID No.3所示，所述GhROP6组成性激活突变蛋白的氨基酸序列如SEQ ID No.4所示；
b、遗传转化：将步骤a得到的载体转化棉花，即得到纤维长度增长的棉花。
4.如权利要求3所述的改良棉花纤维长度的方法，其特征在于：所述小GTP结合蛋白基因GhROP6的核苷酸序列如SEQ ID No.1所示；所述GhROP6组成性激活突变基因的核苷酸序列如SEQ ID No.2所示。
5.如权利要求3或4所述的改良棉花纤维长度的方法，其特征在于：所述的小GTP结合蛋白基因或其组成性结合突变体基因在组成型启动子CaMV35S的介导下表达。
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| WO2008067841A1 (en) * | 2006-12-08 | 2008-06-12 | Swetree Technologies Ab | Plants having improved fiber characteristics and method for making the same |
| CN104611348A (zh) * | 2015-02-16 | 2015-05-13 | 西南大学 | 一种棉花纤维优势表达基因、表达载体和应用及含有该基因的转基因棉花的制备方法 |
| CN108623665A (zh) * | 2018-05-14 | 2018-10-09 | 中国农业大学 | GhHUB2蛋白在调控棉花纤维长度和强度中的应用 |
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| WO2008067841A1 (en) * | 2006-12-08 | 2008-06-12 | Swetree Technologies Ab | Plants having improved fiber characteristics and method for making the same |
| CN104611348A (zh) * | 2015-02-16 | 2015-05-13 | 西南大学 | 一种棉花纤维优势表达基因、表达载体和应用及含有该基因的转基因棉花的制备方法 |
| CN108623665A (zh) * | 2018-05-14 | 2018-10-09 | 中国农业大学 | GhHUB2蛋白在调控棉花纤维长度和强度中的应用 |
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| PREDICTED: rac-like GTP-binding protein ARAC3 [Gossypium raimondii];Blast;《NCBI Genbank》;20150522;ORIGIN * |
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