CN110511932A - 棉花纤维长度相关microRNA477及其前体DNA和应用 - Google Patents
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Abstract
本发明公开了一种棉花纤维长度相关microRNA及编码该棉花纤维长度相关microRNA的前体DNA,所述棉花纤维长度相关microRNA序列为如SEQ ID NO:1所示的核酸序列或由SEQ ID NO:1所示序列经过一个或几个核酸的取代和/或缺失和/或添加且与棉花纤维长度相关的衍生的核酸序列。本发明提供的棉花纤维长度相关microRNA及其前体DNA,将前体DNA导入棉花,可以显著提高棉花纤维长度。本发明对于培育棉花新品种具有重要的意义,适合于推广应用。
Description
技术领域
本发明涉及棉花纤维长度相关microRNA及其前体DNA和应用,属于生物技术领域。
背景技术
我国是世界上棉花种植面积最大,棉花纤维消费量最多的国家。棉花纤维也是一个很好的研究单细胞生长发育的模型(Chen et al. 2007b; Wendel et al. 2003)。世界上95%以上的棉花是由陆地棉产生的,这是因为它具有广泛的适应性和较高的产量,而其余5 %的棉花产量主要来自海岛棉,海岛棉的纤维具有超高的长度、强度以及更细的马克隆值。棉纤维是从棉籽的表皮中分化出来的单细胞,每粒棉籽都含有大约25000个细胞可以发育成纤维细胞(Kim et al. 2001; Lee et al. 2007)。棉纤维长度有的可达到3~5cm。棉纤维的主要成份是纤维素,其含量可以达到95%以上。棉花纤维细胞的这些特性使棉纤维成为研究细胞分化和细胞快速伸长以及研究纤维素生物合成和利用的一个很好的模型。长纤维高比强低马克隆值的棉花纤维是棉花育种的主要目标。
目前棉花纤维发育过程中miRNA的功能研究进展缓慢,尽管一系列的研究已经发现miRNAs在调节纤维发育中的作用,但很少有研究对某一个miNRAs在棉花纤维中的确切功能进行研究。
发明内容
有鉴于此,本发明提供了棉花纤维长度相关microRNA及其前体DNA和应用,具体采用如下技术方案:
棉花纤维长度相关microRNA,获自棉花,命名为MIR477,其序列为如SEQ ID NO :1所示的核酸序列;
或,由SEQ ID NO :1所示序列经过一个或几个核酸的取代和/或缺失和/或添加且与棉花纤维长度相关的衍生的核酸序列。
上述microRNA的核酸序列可人工合成,也可直接在棉花中克隆得到,如通过将SEQID NO :1所示的核酸序列中缺失一个或几个核酸,和/或进行一个或几个碱基对的突变得到。
本发明还提供了编码上述棉花纤维长度相关microRNA的前体DNA,其基因序列为以下的任意一种DNA分子:
(1)编码区如SEQ ID NO :2所示的DNA分子;
(2)与(1)中DNA分子杂交且编码与棉花纤维长度相关microRNA的DNA分子;
(3)与(1)或(2)中DNA分子具有90%以上同源性且编码与棉花纤维长度相关蛋白的DNA分子。
进一步的,(2)与SEQ ID NO :2所示的DNA分子杂交的条件为0.1×SSPE (或 0.1×SSC),0.1%SDS的溶液,在DNA或者RNA 杂交实验中65℃下杂交并洗膜。
本发明还提供了上述前体DNA在制备重组表达载体、表达盒、转基因细胞系或重组菌中的应用。
可用现有的植物表达载体构建含有棉花纤维长度相关microRNA序列的重组表达载体。植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。使用棉花纤维长度相关microRNA构建重组表达载体时,可在其转录起始核苷酸前加上任何一种增强型、组成型、组织特异型或诱导型启动子,它们可单独使用或与其它的植物启动子结合使用;此外,使用棉花纤维长度相关microRNA构建重组表达载体时,还可使用增强子,包括翻译增强子或转录增强子。为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用植物表达载体进行加工,如加入在植物中表达可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。
重组表达载体具体可为将pBI121载体的BamHI和SacI酶切位点间的片段替换为序列表的序列2自5’端第1-95位所示的DNA分子得到的重组表达载体;
或,重组表达载体具体可为将pCLCrV-A棉花曲叶病毒载体的SpeI和AscI酶切位点间的片段替换为序列表的序列2自5’端第1-95位所示的DNA分子得到的重组表达载体。
本发明还提供了上述棉花纤维长度相关microRNA,或,上述前体DNA调控棉花纤维长度中的方法,步骤为:改变棉花中棉花纤维长度相关microRNA的表达量,得到不同纤维长度的棉花。
方法中,棉花纤维长度相关microRNA序列可以通过上述任一重组表达载体导入棉花。
重组表达载体可通过Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化到棉花细胞或组织中。
本发明提供了棉花纤维长度相关microRNA及其前体DNA,将其前体DNA序列导入棉花,增加棉花中棉花纤维长度相关microRNA的表达量,可以显著提高棉花纤维长度。本发明对于培育棉花新品种具有重要的意义,适合于推广应用。
本发明还提供了上述棉花纤维长度相关microRNA,或,上述前体DNA,或,上述方法在植物育种中的应用。
进一步,植物为锦葵科棉属植物,包括陆地棉、海岛棉、亚洲棉。
附图说明
图1为本发明实施例中棉花曲叶病毒载体侵染的棉花植株中MIR477的qRT-PCR检测结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
在以下实施例中,pCLCrV载体为文献Gu Zhouhang,Huang Changjun,Li Fangfanget al. A versatile system for functional analysis of genes and microRNAs incotton.[J] .Plant Biotechnol. J., 2014, 12: 638-49中所涉及的pCLCrV载体;
农杆菌GV3101(郑州尚医生物科技有限公司,华越洋品牌);
棉花品种:中棉所24(中国农业科学院棉花研究所);
其他未做特殊说明的实验方法,均为本领域内常用的实验方法;未做特殊说明的材料,均为本领域内常用的材料,以下实施例中所进行的定量实验,均设置三次重复实验,结果取平均值。
实施例1
MIR477成熟序列及其前体序列miR477b的获得
提取棉花陆海回交近交系“Long”和“Short”开花后10天的纤维中small RNA,并进行miRNA高通量测序。经过大量序列分析、表达量分析与功能验证,从测序结果中发现了一个miRNA的表达量与纤维长度成显著正相关。其成熟序列如序列表的序列1所示,能产生该miRNA的前体序列如序列表的序列2所示。
将序列表的序列1所示的成熟miRNA命名为MIR477。将能产生该miRNA的前体序列命名为miR477b。
实施例2
成熟MIR477及其前体miR477b的功能验证
一、病毒诱导过表达植株的获得
1、重组表达载体的构建:将pCLCrV-A载体的SpeI和AscI酶切位点间的片段替换为序列表的序列2自5’端第1-95位所示的DNA分子,得到重组表达载体pCLCrV-A::miR477b并进行测序验证。
2、将步骤1得到的重组表达载体pCLCrV-A:: miR477b导入农杆菌GV3101,得到重组菌GV3101::miR477b。
3、将步骤2得到的重组菌pCLCrV-A::miR477b通过蘸花法转入受体棉花品种中棉所24中,按照以下步骤进行:
(1)将pCLCrV-A::miR477b农杆菌和辅助转化pCLCrV-B农杆菌菌液分别在含有利福平、链霉素和卡那霉素的抗性LB液体培养基中进行扩繁,扩繁条件:28℃,190rpm,培养大概16h。扩繁至菌液活性OD600在1.5到2.0之间;
(2)4000rpm对菌液进行离心,10min,使用转化介质对离心后的菌体进行重新悬浮,调节OD600在1.5左右;
(3)重新悬浮后的菌液在25℃避光静置3h及以上时间;
(4)将pCLCrV-B菌液分别与pCLCrV-A空载体、pCLCrV-阳性对照、pCLCrV-A::miR477b连有目的基因的菌液按照等体积混合均匀;
(5)将棉花种子进行浸种、育苗,在子叶即将展平时进行菌液侵染。用注射器针头在棉花子叶背面划出伤口,然后将混匀的菌液注射到子叶中;
(6)注射后的棉花植株黑暗处理24h,之后在25℃的温室进行培养;
(7)阳性对照表现出叶片白化表型后对所有注射的棉花植株摘取幼嫩叶片,使用TPS裂解液进行裂解并进行PCR扩增,鉴定阳性植株并移苗到棉花温室。
转化介质配制的原料及用量参照如下表1:
表1转化介质原料及用量
药品名称 | 工作液浓度 |
MgCl<sub>2</sub> | 10 mM |
MES 2-(4-Morpholino)ethanesulfonic acid | 10 mM |
AS acetosyringone(DMSO溶解) | 100 μM |
二、病毒空载体植株的获得
采用pCLCrV-A空载体替代重组表达载体pCLCrV-A::miR477b,按照步骤3-4进行操作,得到转病毒空载体株系。
三、转病毒载体植株的qRT-PCR检测
待测植株:中棉所24(WT)、病毒诱导过表达株系(V1-V3)的植株、转病毒空载体株系植株。
1、提取待测棉花开花后10天的纤维中的small RNA。
2、以步骤1得到的RNA为模板,并利用颈环颈环引物RT-MIR477将MIR477反转录为cDNA。采用引物qRT-MIR477-F和引物qRT-MIR477-R组成的引物对进行荧光定量PCR,检测待测植株中成熟miRNA:MIR477的表达情况;采用引物Ghubq6-F和引物Ghubq6-R组成的引物对检测内参基因UBQ6。
RT-MIR477:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAG GAAGC(5’-3’);
qRT-MIR477-F:TCTCCCTCAAGGGCTTCC(5’-3’);
qRT-MIR477-R:GTGCAGGGTCCGAGGTATTC(5’-3’);
Ghubq6-F:CATTTCTCGATTTGTGCGTGTC(5’-3’);
Ghubq6-R:GGGGACATCCGATAAAATTGG(5’-3’)。
结果如图1所示。结果表明,相对于中棉所24(WT),三个病毒过表达株系V1-V3中MIR477都正常超量表达。转空载体株系中MIR477的表达情况与中棉所24(WT)相同。
四、功能鉴定
待测植株:中棉所24(WT)、病毒诱导过表达株系(V1-V3)的植株、转病毒空载体株系植株。
成熟纤维长度测量
在安阳省中国农业农村部下属棉花纤维品质测试中心采用使用高级纤维信息系统(AFIS)以单铃为单位进行纤维长度的测量,每个植株测量不少于3个单铃。
结果如表2所示。
表2成熟纤维长度测定结果
结果表明,病毒诱导过表达株系相对于中棉所24(WT),成熟纤维长度存在显著差异。空载体株系成熟纤维长度与中棉所24(WT)无差异。
上述结果说明:本发明的microRNA:MIR477具有调节成熟纤维长度的功能。
序列表
<110> 中国农业科学院棉花研究所
<120> 棉花纤维长度相关microRNA477及其前体DNA和应用
<141> 2019-07-17
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
cactctccct caagggcttc c 21
<210> 2
<211> 95
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctcatttccc actctccctc aagggcttcc gtatttgctt accttttttt taagggcttt 60
aaaatgaatc ccttggggag agagtggaca atgtt 95
Claims (7)
1.棉花纤维长度相关microRNA,其特征在于,所述棉花纤维长度相关microRNA序列为如SEQ ID NO :1所示的核酸序列;
或,由SEQ ID NO :1所示序列经过一个或几个核酸的取代和/或缺失和/或添加且与棉花纤维长度相关的衍生的核酸序列。
2.编码权利要求1所述棉花纤维长度相关microRNA的前体DNA。
3.根据权利要求2所述的一种编码权利要求1所述棉花纤维长度相关microRNA的前体DNA,其特征在于,所述前体DNA的基因序列为以下的任意一种DNA分子:
(1)编码区如SEQ ID NO :2所示的DNA分子;
(2)与(1)中DNA分子杂交且编码与棉花纤维长度相关microRNA的DNA分子;
(3)与(1)或(2)中DNA分子具有90%以上同源性且编码与棉花纤维长度相关蛋白的DNA分子。
4.根据权利要求3所述的一种编码权利要求1所述棉花纤维长度相关microRNA的前体DNA,其特征在于,(2)中所述杂交的条件为0.1×SSPE,0.1%SDS的溶液,在DNA或者RNA 杂交实验中65℃下杂交并洗膜;
或,0.1×SSC,0.1%SDS的溶液,在DNA或者RNA 杂交实验中65℃下杂交并洗膜。
5.权利要求2-4任一项中所述前体DNA在制备重组表达载体、表达盒、转基因细胞系或重组菌中的应用。
6.权利要求1所述棉花纤维长度相关microRNA,或,权利要求2-4任一项中所述前体DNA调控棉花纤维长度中的方法,其特征在于,步骤为:改变棉花中棉花纤维长度相关microRNA的表达量,得到不同纤维长度的棉花。
7.权利要求1所述棉花纤维长度相关microRNA,或,权利要求2-4任一项中所述前体DNA,或,权利要求6所述方法在植物育种中的应用。
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CN101812463A (zh) * | 2010-03-09 | 2010-08-25 | 南京农业大学 | 陆地棉中一个新的纤维发育相关microRNA基因 |
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