CN116622725A - 杂交鹅掌楸LhMFT2基因及应用 - Google Patents
杂交鹅掌楸LhMFT2基因及应用 Download PDFInfo
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Abstract
本发明公开了一种杂交鹅掌楸LhMFT2基因,属于基因工程技术领域,所述基因的核苷酸序列如SEQ ID NO.1所示,将杂交鹅掌楸LhMFT2基因转入野生型拟南芥中,可以延长拟南芥植株的童期,提高产量且抗倒伏,在分子育种中具有重要应用价值。
Description
技术领域
本发明涉及基因工程技术领域,更具体地说是涉及杂交鹅掌楸LhMFT2基因及应用。
背景技术
鹅掌楸属(Liriodendron L.)隶属于木兰科,为多年生温带落叶树种。该属树种为高大乔木,树干通直,材质优良,花叶俱美,少病虫害、抗污染能力强,具有较高的观赏价值。鹅掌楸属种间杂交后代—杂交鹅掌楸(L.chinense Sarg.×L tulipifera L.)则具有明显的杂种优势,与亲本相比具有更强的速生性和抗逆性,山地大面积造林胸径年生长量可达2.0cm以上,是良好的材用树种和碳汇树种,具有极强的开发利用前景。因此,鹅掌楸属树种尤其是其种间杂种乃极少数集较好速生性、优质性、适应性等优良性状于一身的树种之一,数十年的栽培试验证实了其多方面明显的杂种优势。
磷脂酰乙醇胺结合蛋白(PEBP)广泛存在于动物、植物和微生物中。植物PEBP基因,充当各种信号通路的调节剂来控制生长和分化,参与开花转换和营养生长。该家族分有三个主要分支:FT-like,TFL-like和MFT-lik基因。其中,FT-like和TFL-like研究较为透彻,相关基因主要参与植物的开花和物候调节。MFT亚家族是FT和TFL1亚家族的祖先基因,与FT和TFL相比,MFT的功能研究还不够确切。
因此,如何克隆出一种MFT家族的基因,并对其功能进行研究和应用是本领域技术人员亟需解决的技术问题。
发明内容
有鉴于此,本发明提供了一种杂交鹅掌楸LhMFT2基因及应用。
为了实现上述目的,本发明采用如下技术方案:
一种杂交鹅掌楸LhMFT2基因,所述基因的CDS核苷酸序列如SEQ ID NO.1所示。
一种杂交鹅掌楸LhMFT2蛋白,由SEQ ID NO.1所示核苷酸序列编码而成,蛋白序列如SEQ ID NO.2所示。
权利要求1所述的LhMFT2基因在延长观赏植物童期、推迟植物花期、提高观赏植物花序数量和荚果数量、提高作物产量中的应用。
一种扩增所述LhMFT2基因的引物,所述引物序列如SEQ ID NO.3~SEQ ID NO.4所示;
上游引物LhMFT2-F1:5’-ATGGCTCGCTCTGTGGAGCCTTTGG-3’,如SEQ ID NO.3所示;
下游引物LhMFT2-R1:5’-ATGGCAACCGGCAATTGC-3’,如SEQ ID NO.4所示。
一种植物表达载体pBWA(V)BS:LhMFT2,所述植物表达载体由pBWA(V)BS载体和LhMFT2基因连接而成。
一种植物表达载体pBWA(V)BS:LhMFT2的构建方法,过程包括:先合成:LhMFT2基因的cDNA序列,然后扩增编码区序列,得目标序列;最后将目标序列与pBWA(V)BS载体连接,得植物表达载体。
优选地,以SEQ ID NO.3~SEQ ID NO.4所示的引物扩增编码区序列。
所述的LhMFT2基因在制备转基因微生物中的应用,所述微生物包括农杆菌。
所述的LhMFT2基因在植物育种中的应用。
优选地,所述植物育种包括选育开花数量多的观赏植物品种、抗倒伏能力强和产量高的植物品种。
经由上述的技术方案可知,与现有技术相比,本发明通过挖掘鹅掌楸基因组数据库中的PEBP基因家族序列Lch25487。利用基因同源性,设计引物,成功的在具有杂种优势的杂交鹅掌楸中克隆出了同源基因,通过结构域和数据库比对,证实为PEBP基因家族的MFT基因,命名为LhMFT2。通过对克隆到的LhMFT2基因构建过表达载体,转化拟南芥,多代种植,获得T2代种子;将转基因拟南芥T2代种植和野生型拟南芥在适宜条件下共同培养,表型观察发现转基因植株,花期均有不同程度的推迟,花序变粗变长,且不易倒伏。因此,杂交鹅掌楸LhMFT2基因可以延长拟南芥植株的童期,提高产量且抗倒伏,在分子育种中具有重要应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,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,还可以根据提供的附图获得其他的附图。
图1附图为本发明LcMFT2基因CDS全长的PCR产物的1%琼脂糖凝胶电泳图;
图2附图为本发明构建pBWA(V)BS:LcMFT2表达载体图;
图3附图为本发明转LcMFT2基因拟南芥PCR阳性检测验证图;
图4附图为本发明转LcMFT2基因拟南芥和野生型幼苗花序及开花情况;CK为对照,M-1为LhMFT1基因超表达植株对照,M-2为本发明LhMFT2基因超表达植株对照;
图5附图为本发明转LcMFT2基因拟南芥和野生型花序及结果情况图;左边为野生型拟南芥对照、中间及右边均为LhMFT2过表达植物植株。
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。
实施例中用到的植物材料杂交鹅掌楸取材于江西省南昌市江西省科学院内,于2020年5月取春季幼嫩花枝的花叶混合组织,用液氮冻存后-80℃保存备用。
实施例1LhMFT2基因的挖掘、克隆及表达载体构建
1)杂交鹅掌楸花叶混合总RNA提取
取新鲜的花芽及叶片混合组织1g,于液氮中迅速研磨成粉,用植物RNA提取试剂盒,按照说明书提取总RNA,并用1%琼脂糖凝胶电泳检测,-80℃超低温冰箱中保存待使用。
2)LhMFT2基因引物设计:
下载模式植物拟南芥(https://www.arabidopsis.org)的pebp基因家族氨基酸序列。下载鹅掌楸基因组数据(https://www.ncbi.nlm.nih.gov/),基于拟南芥PEBP基因家族的参考序列,利用TBtools软件,获得鹅掌楸基因组中相关序列19条,再基于保守结构域进行进一步筛选(https://www.ncbi.nlm.nih.gov/Struc ture/bwrpsb/bwrpsb.cgi),最终筛选的到5条还有保守结构域的鹅掌楸PEBP基因。其中,Lch25487基因预测为MFT基因。
以鹅掌楸基因组DNA序列为模板,拓展Lch25487基因及其上下游200bp的区域,设计包含CDS区域的引物序列,引物序列如下:
LhMFT2-F1:5’-ATGGCTCGCTCTGTGGAGCCTTTGG-3’,如SEQ ID NO.3所示;
LhMFT2-R1:5’-ATGGCAACCGGCAATTGC-3’,如SEQ ID NO.4所示。
3)LhMFT2基因的克隆
杂交鹅掌楸cDNA第一链的合成借助利用全式金的反转录试剂盒来完成,并按照试剂盒建议的体系和反应条件进行。反应结束后,利用设计的CDS区域附近基因特异的PCR引物(LhMFT1-F1和LhMFT1-R1)进行扩增。PCR扩增后的产物1.5%浓度的琼脂糖上进行检测,检测是否为目的长度片段,获得目标位置500bp左右的单一条带(图1)。
切取琼脂糖凝胶上的目标产物,使用Takara公司胶回收试剂盒回收目的片段。将回收片段连接入T载体(pMD18T载体),利用T4连接酶进行连接。将10ul连接产物转化至100ul大肠杆菌感受态细胞中。在LB液体培养基上培养后,离心取菌液涂于LB固体培养基进行蓝白斑筛选,并挑取白斑单克隆利用通用M13引物进行菌落PCR。
将PCR产物送上海生工进行测序。将测序结果序列使用NCBI数据库的BALST及CDD数据库(https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi)上进行比对,目标片段均预测为MFT基因。最终确认目标产物为杂交鹅掌楸的MFT基因,命名为LhMFT2,基因CDS区域序列为:
ATGGCTCGCTCTGTGGAGCCTTTGGCCGTAGGCAGAGTCATTGGAGACGTACTCGACATGTTCATCCCGACCGTTGGATTGGTGGTTCAGTACGGATCCAAGCAGGTCACCAATGGCTGTCAGATCAAGCCATCCGCTACCGTCGATCGGCCCAGGGTCCAAATCTACGGTAATCGTCTCTCGAACCATCTCTACACATTGGTGATGACGGACCCTGACGCTCCAAGCCCCAGCGAGCCCACTATGAGGGAATGGCTGCATTGGATCGTTGTAGACATACCAGGAGAGTCTGACGTCACCAAAGGTAAAGAGCTGGTGCCGTACATGGGACCTCGTCCGCCAACCGGCATTCACCGGTACGTCTTCGCTCTATTTCAACAGAAGGAACCGGCAGAGGGGAATCGACCGCCAGATGTCCGAGGAAACTTCAACACCCGCAAGTTCGCGGAGCAAAATGGGCTGGGCCTGCCGGTTGCAGCGGTCTATTTCAACGCACAGAAGGAACCGGTGTCTAAGAAACGCTGA,如SE Q ID NO.1所示;
蛋白序列为:
MARSVEPLAVGRVIGDVLDMFIPTVGLVVQYGSKQVTNGCQIKPSATVDRPRVQIYGNRLSNHLYTLVMTDPDAPSPSEPTMREWLHWIVVDIPGESDVTKGKELVPYMGPRPPTGIHRYVFALFQQKEPAEGNRPPDVRGNFNTRKFAEQNGLGLPVAAVYFNAQKEPVSKKR,如SEQ ID NO.2所示。
4)杂交鹅掌楸LhMFT2基因植物表达载体的构建
将上述获得的包含有杂交鹅掌楸的LhMFT2基因的目标片段菌液进行质粒提取。选择表达载体pBWA(V)BS载体,并对载体和质粒进行酶切反应。将回收片段酶切产物和载体酶切物合并,并用PCR纯化试剂盒纯化,用于下一步的连接反应。利用T4 DNA连接酶进行载体与目标片段链接。将连接产物转化大肠杆菌DH5α感受态细胞,操作步骤按常规的转化步骤即可。完成后,菌液涂于含有卡纳霉素的LB固体培养基,挑取单克隆,进行菌斑PCR鉴定。挑取5个阳性条带对应菌液进行测序,其余菌液接种到含有10mL卡纳霉素抗性的LB培养基中,试管摇菌。最后取对应测序正确的菌液,取一管提取质粒,即获得含有目的片段的pBWA(V)BS:LhMFT2表达载体(图2),保存备用。
实施例2农杆菌介导遗传转化拟南芥及功能分析
1)将野生型拟南芥种子(哥伦比亚拟南芥,Columbia,Col-0)播种于事先浇透水的营养土中(有机质:蛭石:珍珠岩=2:1:1),覆盖保鲜膜保湿,待种子萌发后,揭去薄膜,人工气候室的培养环境为24℃,光照时间为14h,培养至开花。
2)将PCR检测到的含有目的片段的pBWA(V)BS:LhMFT2表达载体的阳性质粒1μL加入到-80℃保存50μL的EHA105农杆菌感受态细胞中,转化后加入1mL LB液体培养基,于30℃、180rpm摇床振荡培养30min进行活化,活化好的农杆菌置于LB固体培养基上培养。从平板培养基上挑取单菌落,再接种到含有Basta抗生素的液体培养基中,在28℃的振荡培养箱中培养16h,使用能扩增LhMFT2基因CDS全长的引物(LhMFT2-F1和LhMFT 2-R1)进行菌液PCR鉴定。检测到的阳性菌液送至上海生工进行Sanger测序确认,确认后的阳性克隆保存于4℃备用。挑取上述测序完全正确的阳性农杆菌,制备重悬液,培养至OD600=0.7。利用悬浮溶液侵染盛开的拟南芥的花序2-3s,并用保鲜膜覆盖,密封避光保持24h后,此操作共计重复3次,每次间隔7天。完成侵染后,正常条件下培养至成熟期,收获拟南芥T0代种子,放于冰箱冷藏7-14天进行春化。
3)转基因植株的筛选
随机取50粒左右的T0代种子均匀撒播于事先浇透水的营养土中(有机质:蛭石:珍珠岩=2:1:1),人工气候室的培养环境为24℃,光照时间为14h。播种后覆盖保鲜膜保湿,待种子萌发后,揭去薄膜,待种子出现两片真叶时,对准幼苗喷洒浓度为40μg/mL的BASTA除草剂,每隔2天喷一次,连续喷3次,除去假阳性植株。
4)转基因植株的PCR鉴定
播种2周后,摘取转基因拟南芥的幼嫩叶片,使用CTAB法提取植物总DNA。以LhMFT2-F1和LhMFT2-R1为引物进行PCR检测。扩增产物在1.5%浓度的琼脂糖上进行检测。共检测8株转基因拟南芥,检测的全部的转基因植株均扩增出目标长度的产物片段,检测目标转基因阳性率为100%;结果见图3。
实施例3LhMFT2基因的转基因拟南芥表型观察
将T1代PCR检测为阳性的转基因拟南芥植株继续培养至成熟期,收获种子得到T2代。待T2代种子干燥后,置于4℃冰箱中春化14天。将春化后T2代种子均匀撒播于于事先浇透水的营养土中(有机质:蛭石:珍珠岩=2:1:1)。培养环境为在24℃、16h光照/8h黑暗的人工气候室。覆保鲜膜培养3天后,撤去保鲜膜,待种子长出2片真叶时,对准转基因植株喷洒浓度为40μg/mL的BASTA除草剂溶液,每隔2-3天喷洒一次,直至长出第四片真叶。继续培养至长出8片真叶,提取叶片DNA并使用其能扩增出全部CDS序列区域的引物(LhMFT2-F1和LhMFT2-R1)进行阳性检测,结果阳性率为100%。继续观察阳性转基因植株与野生型表型差异,见图4和图5。如图4、图5所示,图5中能明显看到CK对照组植株的顶端花絮细弱弯曲,花絮略短,而转基因组花絮直立,花絮长;同理,CK对照组荚果数量明显少于转基因组,即转基因拟南芥花期推迟明显,花序变长、直立性生长更佳,花序数量和荚果数量增多。这一性状如果应用到作物上,可以延长作物童期,增加作物产量和抗倒伏方面具有潜在应用价值。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (10)
1.一种杂交鹅掌楸LhMFT2基因,其特征在于,所述基因的CDS核苷酸序列如SEQ IDNO.1所示。
2.一种杂交鹅掌楸LhMFT2蛋白,其特征在于,由SEQ ID NO.1所示核苷酸序列编码而成,蛋白序列如SEQ ID NO.2所示。
3.权利要求1所述的LhMFT2基因在延长观赏植物童期、推迟植物花期、提高观赏植物花序数量和荚果数量、提高作物产量中的应用。
4.一种扩增所述LhMFT2基因的引物,其特征在于,所述引物序列如SEQ ID NO.3~SEQID NO.4所示;
上游引物LhMFT2-F1:5’-ATGGCTCGCTCTGTGGAGCCTTTGG-3’,如SEQ ID NO.3所示;
下游引物LhMFT2-R1:5’-ATGGCAACCGGCAATTGC-3’,如SEQ ID NO.4所示。
5.一种植物表达载体pBWA(V)BS:LhMFT2,其特征在于,所述植物表达载体由pBWA(V)BS载体和LhMFT2基因连接而成。
6.一种植物表达载体pBWA(V)BS:LhMFT2的构建方法,其特征在于,过程包括:先合成:LhMFT2基因的cDNA序列,然后扩增编码区序列,得目标序列;最后将目标序列与pBWA(V)BS载体连接,得植物表达载体。
7.根据权利要求6所述的一种植物表达载体pBWA(V)BS:LhMFT2的构建方法,其特征在于,以SEQ ID NO.3~SEQ ID NO.4所示的引物扩增编码区序列。
8.权利要求1所述的LhMFT2基因在制备转基因微生物中的应用,所述微生物包括农杆菌。
9.权利要求1所述的LhMFT2基因在植物育种中的应用。
10.根据权利要求9所述的应用,其特征在于,所述植物育种包括选育开花数量多的观赏植物品种、抗倒伏能力强和产量高的植物品种。
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