CN113789308B - 一种表达载体及其在提高大豆生物量中的应用 - Google Patents
一种表达载体及其在提高大豆生物量中的应用 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明公开了一种表达载体及其在提高大豆生物量中的应用,所述表达载体包含大豆内源脂肪酸去饱和关键酶GmHY1基因;所述关键酶GmHY1核苷酸序列如SEQ ID NO.2所示,编码蛋白的氨基酸序列如SEQ ID NO.3所示。本发明利用增强表达目的基因自身的启动子,可以使目的基因能够在其自身表达的位置精准表达,不会改变其表达的时期,将表达量提高2‑10倍;编码框插入位点之前还有一个增强子,可以使增强表达的效果更加明显,将目的基因的表达量提高数十倍。本发明不仅在增强表达目的基因方面存在较大的优势,使转基因效率更高,阳性率更高。依赖此项技术能够获得长势更好、产量更高、性状更为优良的抗除草剂大豆。
Description
(一)技术领域
本发明涉及一种表达载体及其在提高大豆生物量中的应用。
(二)背景技术
转基因作物自1996年商业化种植以来,其种植面积稳步提升。2018年全球转基 因作物的种植面积达到1.917亿公顷。转基因大豆作为世界上推广时间最早、推广面 积最大的转基因作物,2018年全球转基因大豆种植面积9590万公顷,占大豆总种植面积的78%,占全球转基因作物总种植面积的一半以上。面对种植者和消费者的多元 化需求,世界各大农化企业耗费巨资不断开发复合性状转基因作物。
自中国加入世界贸易组织以来,2001—2015年国内大豆种植面积、大豆产量呈现整体上的“双下降”态势,分别由2001年的1.42亿亩、1540.56万吨下降到2015年的 0.98亿亩、1178.50万吨。从国内市场看,2019年国产大豆产量1810万吨,同比增加 215万吨。从国际市场看,2019年我国大豆进口8851万吨,同比增加48万吨。虽然 自2016年起,国内单位面积产量稳步增长,但是国产非转基因大豆单产约为每亩129 公斤,远低于国外转基因大豆每亩320公斤至400公斤的单产水平。我国是大豆主要 消费国和进口国,大豆对外依存度高。故而提高我国大豆的产量迫在眉睫。
大豆(Glycine max.Merr.)是全球第一大油料作物,大豆是主要食用油和豆粕的原料,我国进口大豆20%加工成豆油,80%加工成豆粕,提高大豆的油产量也对提高大豆品质具有重要意义。2019年我国从巴西进口5767万吨,占比65%;从美国进口 1694万吨,占比19%;从阿根廷进口879万吨,占比10%。目前我国进口的大豆及 其产品中,几乎为转基因大豆。
2020年1月和7月,农业农村部正式下发农业转基因生物安全证书。三款耐除草 剂转基因大豆获批,分别是上海交通大学的SHZD3201以及中国农业科学院的中黄 6106,而北京大北农生物技术有限公司的耐除草剂大豆DBN-09004-6获得国内的进 口安全证书更是有助于其在阿根廷进行商业化种植。根据《农业转基因生物安全管理 条例》及相应配套制度,我国转基因种子审批需经历转基因作物安全评价(中间试验 +环境释放+生产性试验+取得安全证书)以及品种审定(2年左右)。自2019年以来, 转基因相关利好政策频频发布,面对病虫害的压力以及国际贸易的摩擦,随着审批速 度的加快,转基因大豆商业化种植有望逐步在国内展开。
(三)发明内容
本发明目的是提供一种表达载体及其在提高大豆生物量中的应用,将大豆内源脂肪酸去饱和的关键基因及抗除草剂的基因通过转基因的技术一同转入大豆品种中,以 提高大豆的油含量,从而使大豆种子变大,单株粒重增加,以达到增产的目的。除此 之外,还能获得抗除草剂的优良性状。
本发明采用的技术方案是:
本发明提供一种提高大豆生物量的表达载体,所述表达载体包含大豆内源脂肪酸去饱和关键酶GmHY1基因;所述关键酶GmHY1基因核苷酸序列如SEQ ID NO.2所 示,编码蛋白的氨基酸序列如SEQ ID NO.3所示。
进一步,所述表达载体包含启动子pGmHY1和增强子,所述启动子pGmHY1核 苷酸序列如SEQ ID NO.1所示,所述增强子核苷酸序列如SEQ ID NO.4所示。
进一步,所述表达载体包含蛋白融合标签,所述蛋白融合标签核苷酸序列如SEQID NO.6所示。
进一步,所述表达载体还包括筛选标记基因,所述筛选标记基因包括抗除草剂基因,所述抗除草剂基因为草丁膦乙酰转移酶基因,核苷酸序列如SEQ ID NO.5所示。
进一步,所述表达载体T-DNA结构为:抗除草剂基因-启动子P35S-启动子 pGmHY1-增强子-蛋白融合标签-基因GmHY1-终止子;所述P35S为花椰菜花叶病毒 35S启动子,终止子为胭脂碱合成酶基因终止子Tnos,所述抗除草剂基因为草丁膦乙 酰转移酶基因,核苷酸序列如SEQ ID NO.5所示,所述增强子核苷酸序列如SEQ ID NO.4所示,所述蛋白融合标签核苷酸序列如SEQ ID NO.6所示。
上述表达载体以双元载体PTF101-Ω-Flag为基础载体,按如下方法构建:将双元载体PTF101-Ω-Flag用Pst1单酶切开,与启动子pGmHY1进行同源组装,使启动子 重组进Flag标签之前,再将重组完成的质粒以BamH1和Sma1进行双酶切,将酶切 后的质粒与关键酶HY1基因再次进行同源重组,使关键酶HY1基因的编码区序列重 组进Flag标签之后,获得组装完成的表达载体PTF101-GmHY1-OE。
本发明提供一种所述表达载体在构建提高生物量的转基因植物中的应用,所述应用是将所述表达载体采用农杆菌介导法转入大豆中,制备生物量提高的转基因大豆。 本发明所述生物量提高是指植物生长高大且迅速,能显著地提高大豆的种子大小,增 加每个植株的结荚数,从而提高大豆的产量。通过气相色谱法检测种子中的油含量,发现种子的总油含量增加。
本发明还提供一种所述表达载体构建的提高生物量的转基因植物细胞,所述转基因植物细胞是将所述表达载体采用农杆菌介导法转入大豆子叶节细胞中获得的,所述 大豆优选为大豆威廉姆斯82。
本发明是将大豆油脂积累过程中的关键酶GmHY1在大豆品种威廉姆斯82中加 倍表达实现的。GmHY1是在大豆种子中特异性高表达的与脂肪酸去饱和相关的酶(图 1),将GmHY1所编码的基因克隆到双元载体PTF101-TATA-Flag中,转化农杆菌 LBA4404,再侵染大豆子叶节,获得转化的大豆植株。对转化后的植株进行抗除草剂 筛选,然后依次进行PCR、荧光定量PCR及免疫印迹实验等方法,证实得到的确实是转基因植株且已经过量表达。T0代植株自交,得到的T1代继续自交得到T2纯合 植株。对得到的纯合T2代植株进行温室和大田农艺性状考察,发现增加GmHY1拷 贝的转基因大豆生长高大且迅速,能显著地提高大豆的种子大小,增加每个植株的结 荚数,从而提高大豆的产量。通过气相色谱法检测种子中的油含量,发现种子的总油 含量增加。
与现有技术相比,本发明有益效果主要体现在:
本发明所用的表达载体较之于其他载体而言更强大的功能是可以更为便利直接地表达目的基因自身的启动子。相较于对于该技术领域普遍采用的花椰菜花叶病毒(CaMV)的35S启动子启动编码框的表达载体,利用增强表达目的基因自身的启动子, 可以使目的基因能够在其自身表达的位置精准表达,而不是广泛表达;并且也不会改变其表达的时期,故而我们能更加准确地分析该基因的功能。此外,编码框插入位点 之前还有一个增强子,可以使增强表达的效果更加明显。35S启动子启动的基因表达 是一个较强的启动子,通常情况下可以将目的基因的表达提高到10~100倍,但是采 用目的基因的自身启动子启动目的基因往往只能将表达量提高2-10倍。而我们加了增强子之后,即使采用目的基因自身的启动子,仍然可以将目的基因的表达量提高数 十倍。本发明不仅在增强表达目的基因方面存在较大的优势,并且通过技术改良,使 转基因效率更高,阳性率更高。依赖此项技术能够获得长势更好、产量更高、性状更 为优良的抗除草剂大豆。
(四)附图说明
图1、GmHY1基因在威廉姆斯82大豆中的时空表达;(a)大豆不同发育时期种 子样品的示意图;(b)GmHY1在大豆不同组织中的RNA表达量柱形图;(c)GmHY1 在种子不同发育时期的表达量柱形图。
图2、增强表达载体PTF101-GmHY1-OE结构示意图及在种子中的表达水平;(a) 表达载体PTF101-pHY1-OE中T-DNA区域的图谱;LB和RB分别是T-DNA区的左 右边界;P35S-花椰菜花叶病毒35S启动子;Tnos-胭脂碱合成酶基因终止子,BlpR- 草丁膦乙酰转移酶基因,pHY1是GmHY1基因的上游启动子序列(SEQ ID NO.1), gHY1是GmHY1基因的编码区域序列(SEQ ID NO.3);Ω-增强子;Flag-蛋白融合标 签;(b)荧光定量PCR鉴定;(c)GmHY1增强表达株系种子中GmHY1的表达水平, GmHY1-OE#1、GmHY1-OE#2为GmHY1基因增强表达的两个转基因株系,WT为非 转基因对照品种威廉姆斯82。
图3、GmHY1基因增强表达株系与威廉姆斯82长势对照图;(a)萌发后水培5 天,Bar=1cm;(b)萌发后水培15天,Bar=4cm;(c)萌发后土培45天,Bar=5cm; (d)萌发后土培45天的株高。
图4、GmHY1基因增强表达株系的农艺性状;(a)温室土培收获的15粒种子照 片,Bar=1cm;(b)温室土培收获的种子单粒重平均值柱形图;(c)田间种植收获单 株大豆种子的平均总粒重;(d)田间种植收获种子的百粒重柱形图。
图5、GmHY1基因增强表达株系田间收获的GmHY1-OE#1、GmHY1-OE#2及 WT种子的含油量(a)及油脂含量百分比(b)柱形图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、增强表达载体PTF101-GmHY1-OE的构建
(1)克隆GmHY1基因及启动子
GmHY1基因在NCBI(National Center for Biotechnology Information(nih.gov))中的分 类ID为3847。选取GmHY1基因起始密码子ATG上游包含5’UTR在内的1800bp为 启动子序列(SEQ ID NO.1),包含起始密码子ATG和终止密码子TGA在内的GmHY1基因编码框序列(SEQ ID NO.2),氨基酸序列如SEQ ID NO.3所示。
采用TPS提取液(100mM Tris-HCl、10mM EDTA、1M KCl,pH=8.0)提取大豆 威廉姆斯82(WT)的基因组,以此为模板,利用引物pGmHY1-F和引物pGmHY1-R, PCR扩增GmHY1启动子(SEQ ID NO.1);利用引物gGmHY1-F和gGmHY1-R,PCR 扩增GmHY1基因编码框序列(核苷酸序列如SEQ ID NO.2所示,氨基酸序列如SEQ ID NO.3所示)。
PCR程序均为:退火温度58℃,程序为98℃2min;98℃15s,58℃30s,72℃ 2min,34个循环;72℃5min。将PCR产物进行一代测序,与基因组序列进行比对, 序列完全一致后,则可用于下一步实验。
引物如下:
pGmHY1-F:5’-accatgttgacctgcaCAATATATTGTTACTTGTGA-3’
pGmHY1-R:5’-GTCTTGCGAccatggcGACCGTTGGAGATCGTTTTT-3’
gGmHY1-F:5’-caggtcgactctagagATGAACGGCGGCGCTGAGGC-3’
gGmHY1-R:5’-tcggtacccggggatcGCATAATAACCATTATATTC-3’。
SEQ ID NO.1
caatatattgttacttgtgaatgaaaatgtaggttattatcaacgagggttacttctctatatactcattcataaaatcaaactactaccaacatatttgagaact atttatttacctagtttgatgaactttgttgattcatacagaagttattaatgaatgtatattctttcatcaacccttatgtaaaaaataatgtaatcatattatgaagtaaaaaatggatcgatacataaaagaaagcacttgaatattgtatcggaatttattgttgtatttcttgtaatttgttgtatatatatataagagtacaagaatctgt atactaattgcctataattgtctagtctataattgcctataatagaaatcctatacaatgatataattgcctatgatagaaatcctatttaaatataattatgatacgttgcctataattgccgttagtgaggtcaattttagtgccacgtggactatccacgtggcactaaaggatgacgtgacatgacacgtggacgtgtctgatgc cacgtcatttgatgataacaaaatgagtaaatagacaatttagtccctaactttgtacccctgttgcatattagtccctaacttaatgaaaaattcaaaatagtctctatcttttacataagtattgcaaaatagtccttccgttaaattttaaagtaatgttgttagtaagttcaattttagtatcacgtcatttgatgatgatagaatgagt gacttcttcaaatttgatggttttaaaccaattgaggcatatatacgaaaaagaactcacacacacttgcacaaataaaaagaaccaaaaatccacagcaacaaccttatctctgtagctgtcaacaccaatgggcgaggtctgcataaccattctcttttcctcttttttttacttcaattaccatcaatgtatcatcttgggttctg attttttttgtgtgttttgaataggaagagaaaaaaccagaggaaaacaaagtggaggagaaaaaagcaaatgaagaagaaaagaaagaagaagagaaaaaaccagaagaatcaaaagatgacaaggaatccaaggaggaatctgcgctgtcagaaatcgtgctaggcacaacctttgcaatgcatggacacttt aagcatgatttctgacatcttttaagttagggattattttgcaacacttatgcaaaagatagggactattttgaatttttcattaagttagggactaatatacaacaggggtacaaagtcagggactaaattacctatttacttgttttgttatcatcaaatgtcgtggcatcagcacgtccacgtgtcatgccacgtcatcctttaga acttaacggcgttactttaaaacttaacggaaggactattttgcaacacttatacaaagatagggactattttaaatttaatattaaattaggtactaatatataaaatgggtataatctcagagactaaattgtctattcactgtattcgcaattctcataatcttccctgtgcggctgtgcgtgtaaataaccatagagaatcacacc cacatcacatgcaatgcaagactaattacccctttgcatttttttattcttaaaaaaagaaaaaaataggaaaattaccaaaaaagaaaacttcgtagtcctagaaacgtaaaaccaacctcaacctaacatgggaaactccgtcaccactttccctcttccgatttttatatcattcgccacatccattattatgtcgtccgcact taaaaaaaacgatctccaacggtc。
SEQ ID NO.2
atgaacggcggcgctgaggcctccgtcaatcacaggcgcagacaccaagcagcttccgctaacggcgttaagatagcaaacggggccatggcgaa gccgtcctcgacgctctgctacgacgcctcgttcatgaaatggaccgtggcggatgctgtccacgtggcgacgcatcattggatgccgtgcttattcgcattagggcttctcttcttcatggccgtggaatacacgctcctcatggttccgccgtcgtcgccgcctttcgatctgggcttcattgccacgcgttccctccac gcactcctcgagtcatcgccgaatctcaacacgctcttcgccgggctcaatacggtacgtcgtttacactttctccagatctgaacgaatcgggacactttttttttctggtttcggatttcgttgtgggcaattgggagatcgaatttgtttggaggaaatgcaaatgggttggtctcaaaatctgatctttttactgcttttttggtt ttagtattcattttgcattaatgggttttgacatggataggtgtttgtggggatgcaaacgagttatatcttatggacgtggctgattgaaggacgccccagagccacgatttcagcattgttcatgttcacatgccgtggaattttagggtactccacccagctcccattgcctcaggtgattcattacttcatcaaccaaagttt caatttttttcattattttaatctttttttattactacctacccaggattttttataccttcattcaatgacttttaaatgttatcatattttttattgaataacgttgtaaatctttttatagattaaactattcacttgctctctatgtttattctcttttaagttttggtcctttaccaaaaaaaaaaaaactctaaatttagtctgtatgccaagttttatag caatcttatacgaaaagagtcatatctagtgatagcaatggaccaaaacttaactaacatgcaggtttagtgactaaaatctctatttttctgatataggaactaaaaccataacttttttttatacaatgtataaaaattaaactaatttatttatttggtgttgaacagggatttttgggctcgggtgtggatttcccagttgggaacg tgtcgtttttcttgtttttttcggggcatgttgcgggttcagtgattgcttccttggacatgaggaggatgcagaggtgggaactggcttggacttttgatgtgctcaatgttttgcaagctgtgaggttgctgggtacaagaggacattacactattgatttggccgtaggggttggtgctggaattctctttgattctttagctggc aagtacgaagatagcaaaaggaatgctgctctatccacaacccacagagcacaatttgattgcgtcaacaatgtggatatagctaaaaaaattaacaaatga。
SEQ ID NO.3
MNGGAEASVNHRRRHQAASANGVKIANGAMAKPSSTLCYDASFMKWTVADAVHVATHH WMPCLFALGLLFFMAVEYTLLMVPPSSPPFDLGFIATRSLHALLESSPNLNTLFAGLNTVFVGMQTSYILWTWLIEGRPRATISALFMFTCRGILGYSTQLPLPQGFLGSGVDFPVGNVSFFLFFSGHVAG SVIASLDMRRMQRWELAWTFDVLNVLQAVRLLGTRGHYTIDLAVGVGAGILFDSLAGKYEDSKRNAALSTTHRAQFDCVNNVDIAKKINK。
(2)增强子
Ω序列是烟草花叶病毒(TMV)5’端非翻译区的一段序列,起到翻译增强的效果,人工合成。增强子核苷酸序列如SEQ ID NO.4所示。
SEQ ID NO.4
acaattaccaacaacaacaaacaacaaacaacattacaattactatttacaattac。
(3)草丁膦乙酰转移酶基因
抗除草剂基因为草丁膦乙酰转移酶基因,原始载体PTF101上有此元件,故不需 要再额外克隆此片段,其具体核苷酸序列如SEQ ID NO.5所示。
SEQ ID NO.5
atgagcccagaacgacgcccggccgacatccgccgtgccaccgaggcggacatgccggcggtctgcaccatcgtcaac cactacatcgagacaagcacggtcaacttccgtaccgagccgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggcatcgcctacgcgggcccctggaaggcacgcaac gcctacgactggacggccgagtcgaccgtgtacgtctccccccgccaccagcggacgggactgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgagcgtgcgcatgca cgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttctggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtcaccgagatc。
(4)蛋白融合标签
Flag标签是常见的蛋白融合标签之一,其序列较短,人工合成获得,其核苷酸序列如SEQ ID NO.6所示。
SEQ ID NO.6:gactacaaggacgacgatgacaag。
2、构建GmHY1-OE增强表达载体
首先我们以载体PTF101(爱荷花州立大学王侃教授惠赠)为基础载体,在载体 的nos终止子之前的Sma1酶切位点处进行酶切,然后按照增强子+Flag的顺序将上 述合成的片段通过同源重组的方式组装到载体中,构成双元载体PTF101-Ω-Flag。参 照图2中a,将双元载体PTF101-Ω-Flag用Pst1单酶切开,与步骤(1)克隆的启动 子pGmHY1进行同源组装,再将重组完成的质粒以BamH1和Sma1进行双酶切,将 酶切后的质粒与克隆的GmHY1基因编码区序列再次进行同源重组,将目的基因的编 码区片段重组在Flag标签之后,从而获得组装完成的增强表达载体PTF101-GmHY1-OE(图2)。
同源重组过程:采用试剂盒ClonExpressionⅡOne Step Cloning Kit(Vazyme)具体实验操作可见该试剂盒的说明书。重组后的产物全部转入大肠杆菌感受态DH5α (100μL,公司购买),冰上静置30min,42℃水浴45s,加入500μL LB液体培养基, 37℃200rpm 1h,吸取100μL菌液均匀涂在含有50mg/L壮观霉素的LB固体培养基 上,37℃黑暗培养12h。
实施例2、利用冷激法将增强表达载体PTF101-GmHY1-OE转化农杆菌LBA4404
制备农杆菌LBA4404感受态细胞:挑取农杆菌LBA4404单菌落接种于3mL YEP 液体培养基中,220rpm、28℃振荡培养至OD600≈0.5,吸取0.5mL菌液接种于50mL 的YEP液体培养基中,300rpm、30℃振荡培养至OD600≈1.0,冰浴10min;3000g 4℃ 离心10min,弃去上清液,加入50mL 4℃预冷的无菌10%甘油重悬沉淀,然后加入 无菌10%甘油至500mL,再次3000g 4℃离心10min,丢弃上清液;重复以上的洗涤 步骤一次;细胞沉淀重悬于25mL 4℃预冷的无菌10%甘油,3000g 4℃离心5min,弃 去上清液,细胞沉淀重悬于0.5mL 4℃预冷的无菌10%甘油,使细胞中浓度为5×1010细胞/毫升,获得农杆菌感受态细胞,分装100μL/管-80℃冻存。YEP液体培养基组成、 YEP固体培养基组成同实施例3。
在装有农杆菌LBA4404感受态细胞(100μL)的离心管中加入500ng实施例1 构建的PTF101-GmHY1-OE载体质粒,置于冰上5分钟,液氮冷激10s,凝结后置于 37℃水浴锅,水浴5分钟,置于冰上2分钟,然后加入不加抗生素的0.5mL YEP液体 培养基,250rpm 28℃振荡培养3小时,再通过3500rpm 3min离心,将菌体沉淀均匀 涂在含有50mg/L壮观霉素和50mg/L链霉素的YEP固体培养基,28℃培养2天,获 得携带PTF101-GmHY1-OE表达载体的农杆菌。
实施例3、PTF101-GmHY1-OE表达载体转化大豆
1、农杆菌介导PTF101-GmHY1-OE表达载体转化大豆
外植体灭菌:将大豆威廉姆斯82的种子用氯气消毒:60-80粒种子在培养皿中, 向灭菌器中其中倒入100mL灭菌液,在干燥锅中密闭灭菌过夜;
外植体吸胀:将灭菌后的大豆种子在萌发培养基上室温吸胀16小时。
农杆菌活化:将实施例2转化完成的携带PTF101-GmHY1-OE表达载体的农杆菌 接种在YEP固体培养基,28℃培养24h后,再转接至YEP液体培养基,28℃培养24h, 获得活化农杆菌菌液。
农杆菌扩繁:将活化农杆菌菌液以体积浓度0.5%接种量接种至250ml的YEP液 体培养基中,28℃培养16h进行农杆菌扩繁,直到农杆菌的对数生长期,此时农杆菌的侵染活力最佳,浓度适宜,获得扩繁后的农杆菌菌液,菌体浓度为OD600=0.8。
农杆菌侵染:将扩繁后的农杆菌菌液在4000rpm 10min的条件下离心富集菌体,去掉上清,再用等体积侵染液重悬,获得农杆菌侵染液,OD600=0.8。在吸胀的大豆外植体的子叶节制造伤口,用扩繁后的农杆菌侵染液浸没,室温侵染30分钟后,去 除种皮。
共培养:将侵染完成后的大豆外植体放在被共培养液体培养基浸湿的滤纸上,在24℃的黑暗环境中使大豆外植体和农杆菌共培养3~5天。
芽诱导:共培养结束,将过长的胚轴修剪至适宜的长度(0.5cm),转移至固体芽 诱导培养基,在24℃16小时光照/8小时黑暗环境培养14天后,去除非丛生芽,在 芽诱导培养基中继代一次。
茎伸长生根:将形成丛生芽的外植体,去掉子叶以及被草丁膦筛选而死掉的芽,转移到茎伸长培养基中,每两周继代一次,每次在基部创造新的切口,以利于对培养 基中养分的吸收,24℃16小时光照/8小时培养。
生根诱导及移栽:当诱导伸长的茎长度达到5厘米以上时,将它们切下,转移到 生根培养罐中,24℃16小时光照/8小时培养1~2周,直到诱导长出3-5条根;可以 移栽至营养土中,在培养箱(28℃16小时光照/8小时湿度40%)中进行练苗;当转 基因幼苗在培养箱中生长至长出3片三出复叶后,移栽至温室或者自然环境进行培育。
转基因过程用的各种培养基的配方如下:
LB液体培养基组成:5g/L酵母提取物,10g/L胰蛋白胨,10g/L氯化钠,调整 pH值至7.0,溶剂为去离子水。
LB固体培养基是在LB液体培养基中添加12g/L琼脂粉。
YEP液体培养基组成:5g/L酵母提取物,10g/L胰蛋白胨,5g/L氯化钠,调整 pH值至7.0,溶剂为去离子水。
YEP固体培养基是在YEP液体培养基中添加12g/L琼脂粉。
B5大量元素:KNO3 2500mg/L、MgSO4·7H2O2 50mg/L、CaCL2·2H2O150 mg/L、(NH4)2SO4 134mg/L、NaH2PO4·H2O 150mg/L,溶剂为水。
B5微量元素:KI 0.75mg/L、H3BO3 3.0mg/L、MnSO4·4H2O 10mg/L、ZnSO4·7H2O2.0mg/L、Na2MoO4·2H2O 0.25mg/L、CoCl2·6H2O 0.025mg/L、CuSO4·5H2O 0.025 mg/L,溶剂为水。
MS大量元素:NH4NO3 1650mg/L、KNO3 1900mg/L、MgSO4·7H2O 370mg/L、 CaCl2·2H2O440 mg/L、KH2PO4·H2O 170mg/L,溶剂为水。
MS微量元素:KI 0.83mg/L、H3BO3 6.2mg/L、MnSO4·4H2O 22.3mg/L、 ZnSO4·7H2O8.6mg/L、Na2MoO4·2H2O 0.25mg/L、CoCl2·6H2O 0.025mg/L、 CuSO4·5H2O 0.025mg/L,溶剂为水。
B5维生素混合液:肌醇100mg/L、烟酸1.0mg/L、盐酸吡哆醇1.0mg/L、盐酸 硫胺10mg/L,溶剂为水。
B5铁盐:Na-乙二胺四乙酸二钠(Na2-EDTA)37.3mg/L、FeSO4·7H2O 27.8mg/L, 溶剂为水。
灭菌液:100mL 10%漂白水中加入5mL 12N的浓盐酸,其中10%漂白水是指含 有效成分为10%次氯酸钠的漂白水。
侵染液:1/10B5大量元素,1/10B5微量元素,1/10B5维生素混合液,30g/L 蔗糖,3.9g/L 2-(N-吗啉代)乙磺酸(MES),pH 5.4,溶剂为去离子水。高压灭菌冷却 后加入过滤灭菌的赤霉素(GA3,0.25mg/L),乙酰丁香酮40mg/L。
共培养液体培养基:1/10B5大量元素,1/10B5微量元素,1/10B5维生素混合 液,30g/L蔗糖,3.9g/L MES,pH 5.4。高压灭菌后加入抽滤灭菌的GA3(0.25mg/L), 半胱氨酸400mg/L,二硫苏糖醇154.2mg/L,40mg/L乙酰丁香酮。
芽诱导培养基:1×B5大量元素,1×B5微量元素,1×B5维生素混合液,1× B5铁盐,30g/L蔗糖,0.59g/L MES,7g/L琼脂,pH 5.7。以上成分高压灭菌冷却后 加入抽滤灭菌的BAP(2,2-双(4-羟基-3-氨基苯基)丙烷)1.11mg/L,头孢噻肟100 mg/L,草丁膦5mg/L。
茎伸长培养基:1×MS大量盐,1×MS微量元素,1×B5维生素混合液,30g/L 蔗糖,0.59g/L MES,7g/L琼脂,pH 5.7。高压灭菌冷却后加入抽滤灭菌的天冬酰胺 50mg/L,谷氨酰胺50mg/L,生长素(IAA)100μg/L,赤霉素(GA3)500μg/L,玉米素1 mg/L,头孢噻肟100mg/L,草丁膦5mg/L。
生根培养基:1×MS大量元素,1×MS微量元素,1×B5维生素混合液,1×B5 铁盐,30g/L蔗糖,0.59g/L MES,7g/L琼脂,pH 5.4。高压灭菌冷却后加入天冬酰 胺50mg/L,谷氨酰胺50mg/L。
2、GmHY1-OE阳性苗鉴定
草丁膦筛选:将1ml有效成分为10%的草铵膦农药加入1L水中制成0.01%草丁 膦溶液。步骤1获得的大豆幼苗植株长出第一片三出复叶之后,一颗植株选取1-3片叶子,用笔做标记,0.01%草丁膦溶液喷湿叶面即可。2-3天之后,对喷施草丁膦的叶 片进行观察,若为阳性植株,则叶片无变化,若为阴性植株则叶片明显变黄,慢慢坏 死、干枯。
PCR鉴定:取50mg各转基因苗的叶片,加入200μL的TPS溶液,研磨机磨碎, 并65℃水浴进行细胞裂解;12000rpm离心10min,吸取上清液并加入等体积异丙醇 进行DNA沉降;再加入500μL 75%酒精洗去蛋白质等杂质,12000rpm离心10min, 去上清液,最后加水溶解,获得各转基因苗的基因组。取1μL通过上述方法获得的基 因组为模板,上游引物(HY1-id-F)为HY1基因特异引物、下游引物(HY1-id-R) 为载体骨架特异引物进行PCR扩增,同时将非转基因的大豆Williams82基因组DNA 做为阴性对照,水为空白对照。用以上基因特异引物扩增获得的目标PCR产物大小 为500bp;若获得目标条带的对应的转基因苗则为阳性苗(图2)。退火温度52℃,程 序为94℃1min;98℃30s,52℃30s,72℃30s,34个循环;72℃2分钟。
荧光定量PCR鉴定:我们通过荧光定量PCR鉴定出各阳性苗中目的基因的表达 量(图2),选取表达量较高的两个株系用于性状的观察。
首先选取如图1所示的步骤1制备的80-100mg大小的种子,用液氮冷冻,研磨 后加入1ml Trizol(购自Invitrogen公司)迅速混匀,室温放置15min,每2~3分钟 颠倒混匀一次,然后10,000rpm离心5min,吸取上清液转入新离心管,弃去沉淀; 加入和上清液等体积的异丙醇溶液沉降RNA;再用75%乙醇洗去杂质,最后用30μL 0.1%的DEPC水溶解,最终获得转基因苗的总RNA。再用反转录试剂盒(TAKARA) 进行反转录获得转基因苗的cDNA。最后以cDNA为模板,GmHY1的特异性引物HY1-qPCR-F/R进行荧光定量PCR。以大豆的内参基因ACTIN为对照。最后通过计 算获得各转基因苗中目的基因的相对表达量(图2中b)。选取表达量较高的两个株系 GmHY1-OE#1、GmHY1-OE#2用于后续性状观察。
TPS溶液:100mM Tris-HCl,10mM EDTA,1M KCl,溶剂为水,pH=8.0。
HY1-id-F:5’-CTAGGTTGGAATTCGGTTGC-3’
HY1-id-R:5’-cgaattcccgatctagtaac-3’
HY1-qPCR-F:GATTGAAGGACGCCCCAGAG
HY1-qPCR-R:AGCAATCACTGAACCCGCAA
3、GmHY1的表达模式分析
根据大豆种子发育的不同阶段和不同程度,以种子的鲜重为标准将大豆种子发育分成了S1~S6等6个不同的时期(图1中a):S1.40-60mg;S2.80-100mg;S3.150-200mg;S4.250-300mg;S5.330-380mg;S6.380-430mg。首先选取了非转基因威廉姆斯82大 豆的不同组织:根、茎、叶、花、根瘤、荚以及不同时期种子的样品,通过对其进行 总RNA的提取,反转录,荧光定量PCR探究了GmHY1在不同组织,不同种子发育 时期的表达情况,结果见图1中b和c。由此发现,GmHY1在种子中的表达量最高, 并且在S1和S2期的表达最高,此后,随着种子的发育,GmHY1的表达逐渐降低。
4、GmHY1-OE转基因材料农艺性状的统计
步骤2的GmHY1-OE#1、GmHY1-OE#2两个株系的转基因大豆T0代通过不断 自交,直到T2代在目的基因位点表现为纯合的材料,用以后续的实验观察。将非转 基因大豆品种威廉姆斯82(Williams82)作为对照材料,与GmHY1-OE#1、 GmHY1-OE#2转基因大豆在温室(16小时/光照,8小时/黑暗,28℃)中进行水培和 土培,以更加便捷直观地观察植株的长势,同时也在海南试验田中(2020年12月-2021 年3月)播种,期间定期观察植株的生长情况,待种子成熟后进行拷种。土培苗是先 在育苗盘上萌发,播种后6-10天可以将苗移栽到花盆中,每盆3颗苗。水培苗是将 种子先在浸湿的滤纸卷上萌发,萌发6天后,可将苗移栽到1/2Hogland营养液中,注 意水培大豆时需要用气泵补充氧气。如图3所示,无论是水培还是土培GmHY1-OE#1、 GmHY1-OE#2的株高显著高于威廉姆斯82。
GmHY1-OE#1、GmHY1-OE#2的种子也明显大于威廉姆斯82,故而百粒重显著 高于威廉姆斯82。此外每株GmHY1-OE#1、GmHY1-OE#2的单株总粒重、单颗种子 大小及百粒重均要高于威廉姆斯82(图4)。
通过气相色谱法(Agilent,CA,USA,DB-23)检测了田间收获的威廉姆斯82、GmHY1-OE#1、GmHY1-OE#2种子的含油量。GmHY1-OE#1、GmHY1-OE#2种子的 含油量显著上升,威廉姆斯82的含油量为200mg/g上下,占20%,GmHY1-OE#1、 GmHY1-OE#2种子的含油量则在21%~23%(图5)。而我国关于大豆油含量的国家标 准中,23%油含量的大豆可以分为一级高含油量大豆。
气相色谱法检测程序:(1)120℃,5min;(2)升温至190℃,保护12min,升温 速率为4℃/min;(3)将温度提高到210℃并保持10分钟,升温速率为2.5℃/min,载气为氮气。最后,使用280℃气相色谱-火焰离子化检测器(7890A,Agilent,USA)进 行检测。
序列表
<110> 浙江大学
<120> 一种表达载体及其在提高大豆生物量中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1800
<212> DNA
<213> 未知(Unknown)
<400> 1
caatatattg ttacttgtga atgaaaatgt aggttattat caacgagggt tacttctcta 60
tatactcatt cataaaatca aactactacc aacatatttg agaactattt atttacctag 120
tttgatgaac tttgttgatt catacagaag ttattaatga atgtatattc tttcatcaac 180
ccttatgtaa aaaataatgt aatcatatta tgaagtaaaa aatggatcga tacataaaag 240
aaagcacttg aatattgtat cggaatttat tgttgtattt cttgtaattt gttgtatata 300
tatataagag tacaagaatc tgtatactaa ttgcctataa ttgtctagtc tataattgcc 360
tataatagaa atcctataca atgatataat tgcctatgat agaaatccta tttaaatata 420
attatgatac gttgcctata attgccgtta gtgaggtcaa ttttagtgcc acgtggacta 480
tccacgtggc actaaaggat gacgtgacat gacacgtgga cgtgtctgat gccacgtcat 540
ttgatgataa caaaatgagt aaatagacaa tttagtccct aactttgtac ccctgttgca 600
tattagtccc taacttaatg aaaaattcaa aatagtctct atcttttaca taagtattgc 660
aaaatagtcc ttccgttaaa ttttaaagta atgttgttag taagttcaat tttagtatca 720
cgtcatttga tgatgataga atgagtgact tcttcaaatt tgatggtttt aaaccaattg 780
aggcatatat acgaaaaaga actcacacac acttgcacaa ataaaaagaa ccaaaaatcc 840
acagcaacaa ccttatctct gtagctgtca acaccaatgg gcgaggtctg cataaccatt 900
ctcttttcct ctttttttta cttcaattac catcaatgta tcatcttggg ttctgatttt 960
ttttgtgtgt tttgaatagg aagagaaaaa accagaggaa aacaaagtgg aggagaaaaa 1020
agcaaatgaa gaagaaaaga aagaagaaga gaaaaaacca gaagaatcaa aagatgacaa 1080
ggaatccaag gaggaatctg cgctgtcaga aatcgtgcta ggcacaacct ttgcaatgca 1140
tggacacttt aagcatgatt tctgacatct tttaagttag ggattatttt gcaacactta 1200
tgcaaaagat agggactatt ttgaattttt cattaagtta gggactaata tacaacaggg 1260
gtacaaagtc agggactaaa ttacctattt acttgttttg ttatcatcaa atgtcgtggc 1320
atcagcacgt ccacgtgtca tgccacgtca tcctttagaa cttaacggcg ttactttaaa 1380
acttaacgga aggactattt tgcaacactt atacaaagat agggactatt ttaaatttaa 1440
tattaaatta ggtactaata tataaaatgg gtataatctc agagactaaa ttgtctattc 1500
actgtattcg caattctcat aatcttccct gtgcggctgt gcgtgtaaat aaccatagag 1560
aatcacaccc acatcacatg caatgcaaga ctaattaccc ctttgcattt ttttattctt 1620
aaaaaaagaa aaaaatagga aaattaccaa aaaagaaaac ttcgtagtcc tagaaacgta 1680
aaaccaacct caacctaaca tgggaaactc cgtcaccact ttccctcttc cgatttttat 1740
atcattcgcc acatccatta ttatgtcgtc cgcacttaaa aaaaacgatc tccaacggtc 1800
<210> 2
<211> 1463
<212> DNA
<213> 未知(Unknown)
<400> 2
atgaacggcg gcgctgaggc ctccgtcaat cacaggcgca gacaccaagc agcttccgct 60
aacggcgtta agatagcaaa cggggccatg gcgaagccgt cctcgacgct ctgctacgac 120
gcctcgttca tgaaatggac cgtggcggat gctgtccacg tggcgacgca tcattggatg 180
ccgtgcttat tcgcattagg gcttctcttc ttcatggccg tggaatacac gctcctcatg 240
gttccgccgt cgtcgccgcc tttcgatctg ggcttcattg ccacgcgttc cctccacgca 300
ctcctcgagt catcgccgaa tctcaacacg ctcttcgccg ggctcaatac ggtacgtcgt 360
ttacactttc tccagatctg aacgaatcgg gacacttttt ttttctggtt tcggatttcg 420
ttgtgggcaa ttgggagatc gaatttgttt ggaggaaatg caaatgggtt ggtctcaaaa 480
tctgatcttt ttactgcttt tttggtttta gtattcattt tgcattaatg ggttttgaca 540
tggataggtg tttgtgggga tgcaaacgag ttatatctta tggacgtggc tgattgaagg 600
acgccccaga gccacgattt cagcattgtt catgttcaca tgccgtggaa ttttagggta 660
ctccacccag ctcccattgc ctcaggtgat tcattacttc atcaaccaaa gtttcaattt 720
ttttcattat tttaatcttt ttttattact acctacccag gattttttat accttcattc 780
aatgactttt aaatgttatc atatttttta ttgaataacg ttgtaaatct ttttatagat 840
taaactattc acttgctctc tatgtttatt ctcttttaag ttttggtcct ttaccaaaaa 900
aaaaaaaact ctaaatttag tctgtatgcc aagttttata gcaatcttat acgaaaagag 960
tcatatctag tgatagcaat ggaccaaaac ttaactaaca tgcaggttta gtgactaaaa 1020
tctctatttt tctgatatag gaactaaaac cataactttt ttttatacaa tgtataaaaa 1080
ttaaactaat ttatttattt ggtgttgaac agggattttt gggctcgggt gtggatttcc 1140
cagttgggaa cgtgtcgttt ttcttgtttt tttcggggca tgttgcgggt tcagtgattg 1200
cttccttgga catgaggagg atgcagaggt gggaactggc ttggactttt gatgtgctca 1260
atgttttgca agctgtgagg ttgctgggta caagaggaca ttacactatt gatttggccg 1320
taggggttgg tgctggaatt ctctttgatt ctttagctgg caagtacgaa gatagcaaaa 1380
ggaatgctgc tctatccaca acccacagag cacaatttga ttgcgtcaac aatgtggata 1440
tagctaaaaa aattaacaaa tga 1463
<210> 3
<211> 279
<212> PRT
<213> 未知(Unknown)
<400> 3
Met Asn Gly Gly Ala Glu Ala Ser Val Asn His Arg Arg Arg His Gln
1 5 10 15
Ala Ala Ser Ala Asn Gly Val Lys Ile Ala Asn Gly Ala Met Ala Lys
20 25 30
Pro Ser Ser Thr Leu Cys Tyr Asp Ala Ser Phe Met Lys Trp Thr Val
35 40 45
Ala Asp Ala Val His Val Ala Thr His His Trp Met Pro Cys Leu Phe
50 55 60
Ala Leu Gly Leu Leu Phe Phe Met Ala Val Glu Tyr Thr Leu Leu Met
65 70 75 80
Val Pro Pro Ser Ser Pro Pro Phe Asp Leu Gly Phe Ile Ala Thr Arg
85 90 95
Ser Leu His Ala Leu Leu Glu Ser Ser Pro Asn Leu Asn Thr Leu Phe
100 105 110
Ala Gly Leu Asn Thr Val Phe Val Gly Met Gln Thr Ser Tyr Ile Leu
115 120 125
Trp Thr Trp Leu Ile Glu Gly Arg Pro Arg Ala Thr Ile Ser Ala Leu
130 135 140
Phe Met Phe Thr Cys Arg Gly Ile Leu Gly Tyr Ser Thr Gln Leu Pro
145 150 155 160
Leu Pro Gln Gly Phe Leu Gly Ser Gly Val Asp Phe Pro Val Gly Asn
165 170 175
Val Ser Phe Phe Leu Phe Phe Ser Gly His Val Ala Gly Ser Val Ile
180 185 190
Ala Ser Leu Asp Met Arg Arg Met Gln Arg Trp Glu Leu Ala Trp Thr
195 200 205
Phe Asp Val Leu Asn Val Leu Gln Ala Val Arg Leu Leu Gly Thr Arg
210 215 220
Gly His Tyr Thr Ile Asp Leu Ala Val Gly Val Gly Ala Gly Ile Leu
225 230 235 240
Phe Asp Ser Leu Ala Gly Lys Tyr Glu Asp Ser Lys Arg Asn Ala Ala
245 250 255
Leu Ser Thr Thr His Arg Ala Gln Phe Asp Cys Val Asn Asn Val Asp
260 265 270
Ile Ala Lys Lys Ile Asn Lys
275
<210> 4
<211> 56
<212> DNA
<213> 未知(Unknown)
<400> 4
acaattacca acaacaacaa acaacaaaca acattacaat tactatttac aattac 56
<210> 5
<211> 549
<212> DNA
<213> 未知(Unknown)
<400> 5
atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60
gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120
caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180
gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240
aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300
ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360
agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420
ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480
gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540
accgagatc 549
<210> 6
<211> 24
<212> DNA
<213> 未知(Unknown)
<400> 6
gactacaagg acgacgatga caag 24
Claims (8)
1.一种提高大豆生物量的表达载体,其特征在于所述表达载体包含启动子pGmHY1、增强子和大豆内源脂肪酸去饱和关键酶GmHY1基因;所述关键酶GmHY1基因核苷酸序列如SEQID NO.2所示,编码蛋白的氨基酸序列如SEQ ID NO.3所示;所述启动子pGmHY1核苷酸序列如SEQ ID NO.1所示,所述增强子核苷酸序列如SEQ ID NO.4所示。
2.如权利要求1所述的表达载体,其特征在于所述表达载体包含蛋白融合标签,所述蛋白融合标签核苷酸序列如SEQ ID NO.6所示。
3.如权利要求1所述的表达载体,其特征在于所述表达载体还包括筛选标记基因,所述筛选标记基因包括抗除草剂基因。
4.如权利要求3所述的表达载体,其特征在于所述抗除草剂基因为草丁膦乙酰转移酶基因,核苷酸序列如SEQ ID NO.5所示。
5.如权利要求1所述的表达载体,其特征在于所述表达载体T-DNA结构为:抗除草剂基因-启动子P35S -启动子pGmHY1- 增强子-蛋白融合标签-基因GmHY1;所述P35S为花椰菜花叶病毒35S启动子,终止子为胭脂碱合成酶基因终止子Tnos。
6.如权利要求1所述的表达载体,其特征在于所述表达载体以双元载体PTF101-Ω-Flag为基础载体,按如下方法构建:将双元载体PTF101-Ω-Flag用Pst1单酶切开,与启动子pGmHY1进行同源组装,使启动子重组进Flag标签之前,再将重组完成的质粒以BamH1和Sma1进行双酶切,将酶切后的质粒与关键酶GmHY1基因再次进行同源重组,使关键酶GmHY1基因的编码区序列重组进Flag标签之后,获得组装完成的表达载体PTF101-GmHY1-OE。
7.一种权利要求1所述表达载体在构建提高大豆生物量的转基因植物中的应用。
8.如权利要求7所述的应用,其特征在于所述应用是将所述表达载体转入大豆中,制备生物量提高的大豆。
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CN106701784A (zh) * | 2017-01-24 | 2017-05-24 | 河南农业大学 | 大豆油体蛋白基因GmOLEO1及其编码蛋白与应用 |
CN109628464A (zh) * | 2018-12-26 | 2019-04-16 | 浙江大学 | 一种增加大豆产量的方法 |
CN112375782A (zh) * | 2020-11-24 | 2021-02-19 | 河南农业大学 | 一种大豆蛋白激酶基因GmSTK_IRAK的应用 |
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CN106701784A (zh) * | 2017-01-24 | 2017-05-24 | 河南农业大学 | 大豆油体蛋白基因GmOLEO1及其编码蛋白与应用 |
CN109628464A (zh) * | 2018-12-26 | 2019-04-16 | 浙江大学 | 一种增加大豆产量的方法 |
CN112375782A (zh) * | 2020-11-24 | 2021-02-19 | 河南农业大学 | 一种大豆蛋白激酶基因GmSTK_IRAK的应用 |
Non-Patent Citations (1)
Title |
---|
phosphatidylcholine:diacylglycerol cholinephosphotransferase 1 isoform X1 [Glycine max];XP_003528315.1;NCBI;全文 * |
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