CN117004624A - 一种甜橙气孔密度调控基因、编码蛋白及应用 - Google Patents
一种甜橙气孔密度调控基因、编码蛋白及应用 Download PDFInfo
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Abstract
本发明提供了一种甜橙气孔密度调控基因,属于植物基因工程技术领域,所述甜橙气孔密度调控基因命名为CsSDD1,CsSDD1的cDNA核苷酸序列如SEQ I D NO.1所示。该基因能够负向调控柑橘属植物叶片气孔密度,并且,该基因可通过降低叶片气孔密度,提高柑橘属植物对干旱及柑橘溃疡病的抗性。本发明还提供了一种甜橙气孔密度调控基因的编码蛋白及应用。
Description
技术领域
本发明属于植物基因工程技术领域,特别涉及一种甜橙气孔密度调控基因、编码蛋白及应用。
背景技术
柑橘是我国南方广泛栽培的重要果树经济作物。我国柑橘产业大部分集中在南部和东南部地区,分属亚热带季风气候和热带季风气候;夏季高温、降雨不均,冬季气候干燥。我国柑橘栽培多以丘陵山地为主,季节性干旱成为影响柑橘产量最重要的环境因素之一。此外,由地毯草黄单胞杆菌柑橘致病变种(Xanthomonas axonopodis pv.citri,Xac)侵染引起的柑橘溃疡病(citrus bacterial canker disease,CBCD)是世界检疫性柑橘病害,侵染严重时可导致叶片、果实早落,枝稍枯萎,对柑橘产业危害极大。
发明内容
为了解决柑橘属植物对干旱和柑橘溃疡病抗性不足的问题,本发明提供了一种甜橙气孔密度调控基因,命名为CsSDD1,该基因能够负向调控柑橘属植物的叶片气孔密度,并且,该基因可通过降低叶片气孔密度,提高柑橘属植物对干旱及柑橘溃疡病的抗性。
本发明还提供了一种甜橙气孔密度调控基因的编码蛋白及应用。
本发明通过以下技术方案实现:
本发明提供一种甜橙气孔密度调控基因,所述甜橙气孔密度调控基因命名为CsSDD1,CsSDD1的cDNA核苷酸序列如SEQ ID NO.1所示。
基于同一发明构思,本发明提供一种甜橙气孔密度调控基因在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
进一步的,所述柑橘属植物包括柠檬和甜橙。
基于同一发明构思,本发明提供一种甜橙气孔密度调控基因的编码蛋白,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
基于同一发明构思,本发明提供一种甜橙气孔密度调控基因的编码蛋白在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
进一步的,所述柑橘属植物包括柠檬和甜橙。
基于同一发明构思,本发明还提供克隆一种甜橙气孔密度调控基因的引物对。
进一步的,所述引物对包括CsSDD1-F和CsSDD1-R,所述CsSDD1-F的核苷酸序列如SEQ ID NO.3所示,所述CsSDD1-R的核苷酸序列如SEQ ID NO.4所示。
基于同一发明构思,本发明还提供克隆一种甜橙气孔密度调控基因的引物对在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
基于同一发明构思,本发明还提供一种提升柑橘属植物对干旱和柑橘溃疡病抗性的方法,包括以下步骤:
构建CsSDD1基因过表达载体;
将所述CsSDD1基因过表达载体转化至柑橘属植物,筛选获得CsSDD1基因过表达株系;
其中,所述CsSDD1基因的cDNA核苷酸序列如SEQ ID NO.1所示。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明一种甜橙气孔密度调控基因,命名为CsSDD1,该基因克隆自纽荷尔甜橙,能够负向调控柑橘属植物如柠檬的叶片气孔密度,将CsSDD1基因导入柠檬,过表达或超表达CsSDD1基因可通过降低气孔密度提高柠檬对干旱及柑橘溃疡病的双重抗性。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为本发明中甜橙CsSDD1基因克隆与功能鉴定的流程示意图。
图2为实施例2中甜橙CsSDD1基因的亚细胞定位图。
图3为实施例3中过表达CsSDD1基因柠檬植株再生图:图中A为共培养;B为抗性芽发生;C为抗性芽伸长;D为抗性芽生根;E为移栽成活的再生植株。
图4为转甜橙CsSDD1柠檬再生植株阳性鉴定(A)及基因表达图(B):A中M是DNAmarker;1是阳性质粒对照;2是野生型柠檬;3、4、5泳道是转基因阳性柠檬再生植株;A中上方胶图是扩增出的NPTII抗生素标记基因特异片段,A中下方胶图是35S启动子上游引物与CsSDD1基因下游引物配对扩增出的特异片段;B中WT是野生型柠檬;OE6、OE34是三个不同的转基因系;“*”代表p<0.05显著性水平差异;“***”代表p<0.001显著性水平差异。
图5为转甜橙CsSDD1基因柠檬下表皮气孔密度图:A中WT是野生型柠檬;OE6、OE34为两个不同的转基因株系;“***”代表p<0.001显著性水平差异;标尺为80μm。
图6为过表达甜橙CsSDD1基因柠檬叶片离体失水图:A图与B图中CK是野生型柠檬;6号、34号为转基因系。
图7为过表达CsSDD1基因柠檬叶片离体接种柑橘溃疡菌第9d病斑表型及病斑面积图:A与B中WT代表野生型柠檬;6、34号株系代表两个不同的转基因系;“**”代表p<0.01显著性水平差异。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买得到或者可通过现有方法制备得到。
本发明整体思路如下:
气孔是气体出入植物的通道,降低气孔密度有望减少水分散失从而提高作物耐旱能力;此外,气孔也是柑橘溃疡菌入侵的主要通道,降低气孔密度还有望增强柑橘的物理抗性。气孔密度和分布(Stomatal Density and Distribution)调控基因SDD1编码一种枯草杆菌蛋白酶丝氨酸激酶。玉米、拟南芥、菘蓝三种植物SDD1基因均可负调控气孔密度,但果树中尚未进行克隆及功能鉴定。
柑橘童期长,部分柑橘资源如甜橙(Citrus sinensis L.)、枳(Poncirustrifoliata L.)具多胚现象。合子胚的生长能力通常不如珠心胚,因此传统杂交育种方法不仅周期长,多胚型母本常需通过胚挽救才能获得杂种苗;此外,杂交为两性亲本配子组合,杂交后代遗传组成改变较大,难以实现针对个别性状进行改良的目的。随着基因工程技术的日渐成熟,农杆菌介导的遗传转化技术已广泛应用于柑橘遗传改良。
基于以上分析,从柑橘资源甜橙中克隆气孔密度调控基因,通过转基因手段降低柑橘气孔密度,有望达到既增强干旱抗性又提高对柑橘溃疡病物理抗性的双重目的。本发明从‘纽荷尔’脐橙中分离出一个气孔密度调控基因,命名为CsSDD1,该基因能够负向调控柑橘属植物柠檬的叶片气孔密度,并且,该基因可通过降低柠檬叶片气孔密度,提高柠檬对干旱及柑橘溃疡病的抗性。
下面将结合实施例及实验数据对本申请一种甜橙气孔密度调控基因、编码蛋白及应用进行详细说明。
本发明实施例使用的实验材料包括甜橙(‘纽荷尔’脐橙)叶片、‘优力克’柠檬无菌苗、构建CsSDD1基因过表达载体的质粒(pBI121)、农杆菌(EHA105)等。
实施例1
本实施例进行甜橙气孔密度调控基因CsSDD1全长cDNA的克隆。
在柑橘基因组数据库(http://citrus.hzau.edu.cn/)中获得甜橙CsSDD1基因CDS序列,设计全长扩增引物如下:
正向引物:CsSDD1-F:ATGGAAGCCAAATCTCAGCTTC
反向引物:CsSDD1-R:TTACTTCCAAGTTACTGAAATG。
研究材料甜橙种植在国家脐橙工程技术研究中心种质资源铺,取幼嫩叶片液氮速冻后保存于超低温冰箱,采用购自ambion公司的Trizol buffer提取‘纽荷尔’甜橙叶片RNA,具体方法如下:
(1)取出超低温冻存样品,转至预冷研钵中,液氮速冻后迅速研磨成粉末;称取0.1g粉状样品至预先添加有1mL Trizol buffer的无RNase 2mL离心管,涡旋混匀30s左右。
(2)往1.5mL PCR管中加入1mL Trizol试剂,加入液氮研磨的植物组织0.1g;剧烈震荡,室温放置10-15分钟,裂解细胞并使核酸蛋白复合物分离;4℃,12,000g离心10min。
(3)小心转上清至新的离心管,加入等体积的氯仿,剧烈震荡30s,室温静置10分钟;12,000g,4℃离心15分钟。
(4)小心取上层水相于新的EP管中,加入等量的异丙醇,轻轻颠倒混匀几次,室温下静置10分钟;12,000g,4℃离心10分钟,弃上清。
(5)加入1.0mL预冷的无RNA酶75%乙醇洗涤沉淀,涡旋5min,冰浴浸泡30min。
(6)4℃,12000g离心10min,弃上清,短暂离心数秒,用枪头将剩余液体彻底吸掉,置于超净工作台上风干5-10min;50μL DEPC处理过的水溶解沉淀,即得RNA溶液。
(7)Nanodrop超微量分光光度计测定RNA溶液纯度和浓度。OD260/OD280在1.8-2.1之间,OD260/OD230>2时可进行下一步实验。
(8)以提取的RNA为模板,采用cDNA合成试剂盒(thermo scientific)按照说明书方法获得甜橙叶片cDNA。
(9)以甜橙叶片cDNA为模板利用上述引物CsSDD1-F、CsSDD1-R及购自莫纳生物科技有限公司的高保真酶MonApm TM2×MonHI-FI Mix扩增CsSDD1基因CDS序列全长。PCR扩增体系及程序如表1所示。
表1甜橙CsSDD1基因CDS序列全长PCR扩增体系及程序
PCR产物通过1%琼脂糖凝胶电泳分离,使用TIANgel Midi Purification Kit(北京天根)胶回收试剂盒回收凝胶中的目的片段,具体操作见说明书。将获得的胶回收产物与pTOPO-Blunt载体连接,室温条件下(25-30℃)配置反应体系(表2),置于PCR仪中37℃连接10min,随即将连接产物转化大肠杆菌DH5α感受态,涂布于含50mg/L氨苄青霉素LB平板后37℃过夜培养。
表2 pTOPO载体连接反应体系
第二天挑取单克隆,37℃摇床200r/min摇菌扩繁12h后进行菌液PCR鉴定。选取阳性克隆送擎科生物测序(武汉),最终获得甜橙CsSDD1基因CDS全长序列SEQ ID NO.1。
实施例2
甜橙CsSDD1蛋白的亚细胞定位
同源重组法构建pSuper1300-mas-CsSDD1-GFP亚细胞定位载体,热激法将重组载体转入EHA105根癌农杆菌感受态,-80℃保存备用。以pSuper1300-mas-GFP空载为对照,亚细胞定位按照如下进行:
(1)农杆菌菌液划线于YEB固体培养基平板(含50mg/L卡那霉素),28℃倒置培养36-48h直至单菌落出现。
(2)接种环沾取单菌落进一步划线于YEB固体培养基平板(含50mg/L卡那霉素),28℃倒置扩繁培养36-48h。
(3)无菌刀片刮取菌体于MS液体培养基(含10mM MgCl2、100mM乙酰丁香酮)中28℃200rpm悬浮培养2h,所得农杆菌菌液进一步稀释至OD600为0.6—0.8。
(4)将水培5-7d的新鲜洋葱去掉外层3-4层鳞片叶后,内层叶片在70%酒精中消毒8-10min,无菌水清洗3次。无菌条件下手术刀片将内表皮切成边长1cm左右方块状,转至MS液体培养基(含10mM MgCl2、100mM乙酰丁香酮)中备用。
(5)将切好的洋葱内表皮转入(3)中准备好的农杆菌菌液,侵染20min,期间每隔5min轻轻摇动几次。
(6)在无菌滤纸上吸干多余菌液,平铺于MS固体培养基(含10mM MgCl2、100mM乙酰丁香酮),25℃、暗处共培养16h。
(7)共培养结束后将洋葱内表皮转入蒸馏水中清洗、备用,激光共聚焦显微镜下观察、拍照。
瞬时表达结果如图2所示,pSuper1300-mas-GFP空载转化对照洋葱表皮细胞质壁分离前后细胞壁及细胞质均可见绿色荧光;pSuper1300-mas-CsSDD1-GFP转化洋葱表皮细胞质壁分离前后GFP荧光信号均只出现在细胞壁,表明甜橙CsSDD1蛋白定位于细胞壁。
实施例3
如图1,农杆菌介导的CsSDD1基因转化柠檬及植株再生:
一、pBI121-CsSDD1过表达载体构建
采样酶切-连接法构建pBI121-CsSDD1过表达载体,经菌液PCR及双酶切鉴定后导入EHA105农杆菌感受态,选择阳性克隆用于下一步柠檬遗传转化实验。
二、EHA105农杆菌介导CsSDD1基因转化柠檬及转基因植株的获得
1.柠檬无菌苗制备
剥取成熟柠檬果实种子,1M NaOH溶液清洗10min左右去除果胶,自来水清洗3次,每次5min左右。超净台上5%左右安替福民消毒液消毒20min,无菌水换洗3次;去除外种皮后播种于含MT播种培养基试管,26±1℃、暗处培养25d左右即得无菌黄化苗,16h光照/8h黑暗条件下培养5-7d稍转绿即可用于侵染。
2.农杆菌菌液准备
取-80℃保存的农杆菌EHA105菌株在含50mg/L卡那霉素的LB固体培养基(胰化蛋白胨10g/L+酵母提取物5g/L+氯化钠10g/L+琼脂15g/L)上划线,28℃暗处倒置培养36-48h获单菌落;挑取单菌落于新的含50mg/L卡那霉素的LB固体培养基上划线,同样条件培养36-48h,无菌手术刀片刮取所有菌体于不加抗生素的MT液体培养基中,200rpm,28℃振荡培养1.5-2h后调节菌液OD600至0.6-0.8后备用。
3.农杆菌侵染
上胚轴切段浸泡于含20mg/L乙酰丁香酮的农杆菌菌液中侵染20min,期间摇动数次;侵染结束后将上胚轴转至无菌滤纸上吸干残余菌液,于垫有灭菌滤纸的MT共生培养基上,21℃暗处培养3d。
4.抗性芽诱导
将共培养结束的上胚轴切段转入锥形瓶中,无菌水漂洗3次,每次约5min,无菌滤纸吸干上胚轴残余菌液后转至含400mg/L头孢霉素、50mg/L卡那霉素的MT生芽培养基上,26±1℃暗处培养7d,之后转入16h光照/8h黑暗条件下诱导切口部位抗性芽发生。
5.植株再生
抗性不定芽生长至1cm左右时转入MT伸长培养基,每20d继带一次;待茎段伸长至2cm左右时从基部将抗性芽切离后转入生根培养基中生根。不定根生长至试管底部时取出柠檬再生小苗,洗净根部残留培养基,蒸馏水中练苗1w左右转入含灭菌营养土塑料杯中,26±1℃、16h光照/8h黑暗条件下恒温培养箱中生长(如图3)。
6.再生植株DNA提取及PCR鉴定
CTAB法提取再生植株叶片基因组DNA,无菌水稀释至100ng/μL左右,为避免柠檬同源基因干扰,以35S启动子特异序列为正向引物、CsSDD1基因特异序列为反向引物进行阳性植株PCR检测。正向引物35S-CsSDD1-F序列为CTATCCTTCGCAAGACCC,反向引物CsSDD1-R序列为CCTGGCACCAATGAGTTT。对转甜橙CsSDD1基因再生植株柠檬基因组DNA PCR扩增获得与预期大小一致的片段,表明再生植株为阳性(图4)。利用选择标记基因新霉素磷酸转移酶NPTII基因特异引物NPTII-F:ATGACTGGGCACAACAGACAA及NPTII-R:CGATACCGTAAAGCACGAGGA也扩增出了与预期大小一致的条带,进一步佐证了转基因阳性植株的真实性(图4A)。
7.转CsSDD1基因柠檬表达分析
提取转基因阳性再生植株叶片RNA并反转录为cDNA(方法同实施例1),以柑橘β-actin基因为内参,正向引物为β-actin-F:CCGACCGTATGAGCAAGGAAA,反向引物为β-actin-R:TTCCTGTGGACAATGGATGGA,采用qRT-PCR方法对转基因再生植株CsSDD1基因进行表达分析。CsSDD1基因qRT-PCR分析的上、下游特异引物分别为CTGCTGGGACTTCGGTTTCT及TGCCACATCCATTGCAGCTA。结果表明,6号、34号转化系CsSDD1基因表达量量均显著高于对照(图4B)。
8.转CsSDD1基因柠檬再生植株气孔密度测定
以再生的阴性柠檬为对照,对CsSDD1基因表达量显著提高的6号、34号转化系气孔密度进行了测定。取生长节位相同的阳性转基因柠檬再生植株成熟叶片各3片,将无色指甲油均匀涂抹于叶片中部下表皮组织,自然风干30min后,用镊子小心揭起指甲油膜,切成边长为0.5cm左右块状,制备下表皮装片。奥林巴斯显微镜下观察、拍照,利用Imag J软件测定气孔密度。结果表明,转基因株系OE6、OE34的气孔密度(326.2个/mm2、340.9个/mm2)显著低于野生型(393.3个/mm2)(图5)。
实施例4
转基因柠檬离体叶片耐脱水分析
以野生型柠檬为对照,对转甜橙CsSDD1基因阳性转化系柠檬6号(OE6)、34号(OE34)转化系叶片离体失水率变化进行了测定。每个株系各取生长发育一致的3片叶片,背面朝上摆放于实验桌上,室内环境下进行离体失水处理。每隔1h小时称重1次,连续处理6h,实验重复3次。参照公式RW=(W0-Wt)/W0计算离体失水率。W0:干旱处理前叶片重量;Wt:离体失水t小时后叶片重量。
结果表明,离体失水1h以上时(含1h),6号转化系失水率明显低于野生型对照;离体失水2h以上时(含2h),34号转化系叶片离体失水率也显著低于野生型对照(图6)。由此可见,过表达CsSDD1基因导致的气孔密度降低可明显降低叶片离体失水率。
实施例5
转CsSDD1基因柠檬抗溃疡病分析
1.柑橘溃疡菌菌液制备
超低温冰箱取出柑橘溃疡菌菌种(Xanthomonas axonopodis pv.citri,Xac),无菌条件下接种环沾取甘油菌体划线于SPA固体培养基(蔗糖20g L-1,蛋白胨5g L-1,硫酸镁0.25g L-1,磷酸氢二钾0.5g L-1,琼脂8g L-1,pH=7.0)平板,37℃倒置培养36h左右获单菌落,再次挑取单菌落于SPA固体培养基上划线扩繁36h左右,无菌刀片刮取菌体于SPB液体培养基悬浮培养并调整至OD600=0.3后用于接种。
2.离体针刺
以野生型柠檬为对照,取转甜橙CsSDD1基因柠檬6号(OE6)、7号(OE7)、34号(OE34)转化系各3片成熟、健康叶片,75%酒精擦拭消毒后叶片背面朝上摆放于垫有两层无菌水湿润滤纸的培养皿中。接种针五针一束,沾取上述柑橘溃疡菌菌液沿叶片主脉对称针刺接种,每侧5个接种部位;接种结束后保鲜袋包裹培养皿并转入26±1℃恒温培养箱中发病培养。
3.病斑面积测定
发病结束后数码相机拍照,Image J软件测定病斑面积。结果表明,6号(OE6)、34号(OE34)转化系病斑面积均显著小于野生型柠檬,表明过表达甜橙气孔密度调控基因CsSDD1可通过降低气孔密度提高柠檬对柑橘溃疡病的抗性(图7)。
综上所述,本发明通过农杆菌介导的遗传转化方法将CsSDD1基因导入柠檬,获得的转基因植株下表皮气孔密度显著降低,即过表达CsSDD1基因明显降低了柠檬叶片气孔密度,表明该基因具有负调控气孔密度的作用;通过离体失水与离体接种实验表明,获得的转CsSDD1基因柠檬抗旱性与柑橘溃疡病抗性均明显提高。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种甜橙气孔密度调控基因,其特征在于,所述甜橙气孔密度调控基因命名为CsSDD1,CsSDD1的cDNA核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的一种甜橙气孔密度调控基因在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
3.根据权利要求2所述的应用,其特征在于,所述柑橘属植物包括柠檬和甜橙。
4.如权利要求1所述的一种甜橙气孔密度调控基因的编码蛋白,其特征在于,所述编码蛋白的氨基酸序列如SEQ ID NO.2所示。
5.如权利要求4所述的编码蛋白在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
6.根据权利要求5所述的应用,其特征在于,所述柑橘属植物包括柠檬和甜橙。
7.克隆如权利要求1所述的一种甜橙气孔密度调控基因的引物对。
8.根据权利要求7所述的克隆一种甜橙气孔密度调控基因的引物对,其特征在于,所述引物对包括CsSDD1-F和CsSDD1-R,所述CsSDD1-F的核苷酸序列如SEQ ID NO.3所示,所述CsSDD1-R的核苷酸序列如SEQ ID NO.4所示。
9.如权利要求7或8所述的克隆一种甜橙气孔密度调控基因的引物对在提升柑橘属植物对干旱和柑橘溃疡病抗性中的应用。
10.一种提升柑橘属植物对干旱和柑橘溃疡病抗性的方法,其特征在于,包括以下步骤:
构建CsSDD1基因过表达载体;
将所述CsSDD1基因过表达载体转化至柑橘属植物,筛选获得CsSDD1基因过表达株系;
其中,所述CsSDD1基因的cDNA核苷酸序列如SEQ ID NO.1所示。
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