CN114277014B - 拟南芥at5g10290基因在调控植物生长中的应用 - Google Patents
拟南芥at5g10290基因在调控植物生长中的应用 Download PDFInfo
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- CN114277014B CN114277014B CN202111607067.8A CN202111607067A CN114277014B CN 114277014 B CN114277014 B CN 114277014B CN 202111607067 A CN202111607067 A CN 202111607067A CN 114277014 B CN114277014 B CN 114277014B
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Abstract
本发明涉及基因工程技术领域,特别是涉及拟南芥AT5G10290基因在调控植物生长中的应用。本发明所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。本发明所述拟南芥AT5G10290基因在延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力具有较好的生物学功能。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及拟南芥AT5G10290基因在调控植物生长中的应用。
背景技术
在生长和发育过程中,植物体不断接受外界环境因子及本身体内各种信号分子,在体内这类因子和信号分子引发级联反应,最终作出相应的生物学反应使得植物正常生长。首先,膜受体感知外界信号并将其跨膜转化为胞内信号,再通过胞内一系列信号级联反应调控目的基因的表达,引起相应的细胞生理、生化反应。多细胞有机体的生长、发育依赖于机体内、外信号精密的传感机制。受体蛋白激酶作为重要的分子元件与细胞信号转导有着密切的关系。富含亮氨酸重复类受体蛋白激酶(LRR-RLKs)是植物类受体蛋白激酶家族中最大的家族,具有至少223个成员。
LRR-RLKs是一类单次跨膜蛋白,其基本机构包括含有数量不一的LRR单元的胞外结构域,跨膜结构域及参与信号激活的胞内激酶结构域。受体蛋白激酶的胞外域负责感知并结合多种特异的胞外信号分子,进而激活胞内激酶结构域的磯酸化活性,然后通过一系列信号级联反应调节相应基因的表达,调控多种生物学过程。部分LRR-RLKs的功能已经报道,如BRI1/BAK1介导油菜素内酯的信号途径;CLV1参与植物茎端分生组织和花器官的产生;拟南芥中2种LRR-RLKs中的FEI1和FEI2在调控细胞壁的合成进而调节根、花丝以及黄化幼苗下胚轴的细胞生长过程中发挥重要作用;FLS2参与免疫防御反应;水稻中OsSIK1参与植物抗旱。拟南芥中的AT5G10290基因编码蛋白虽然属于LRR-RLKs,但是该蛋白在植物生长中的调控功能尚未证明。
发明内容
为了解决上述问题,本发明提供了拟南芥AT5G10290基因在调控植物生长中的应用。所述拟南芥AT5G10290基因在延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力具有较好的生物学功能。
为了实现上述目的,本发明提供如下技术方案:
本发明提供了拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在调控植物营养生长中的应用;所述调控植物营养生长包括延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力中的一种或多种;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。
优选的,所述提高植物叶片品质包括提高叶片厚度和面积。
优选的,所述提高植物抗倒伏能力包括降低植物株高。
优选的,所述拟南芥AT5G10290基因的核苷酸序列如SEQ ID No.2所示。
优选的,所述包含拟南芥AT5G10290基因的表达载体的基础载体包括pEGAD。
优选的,所述包含拟南芥AT5G10290基因的表达载体的构建方法,包括将所述拟南芥AT5G10290基因***pEGAD的EcoRI/SmaI多克隆位点处。
优选的,所述植物包括十字花科植物。
优选的,所述十字花科植物包括油菜或拟南芥。
本发明还提供了拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在培育具有营养生长期长、叶片品质好和抗倒伏能力强中的一种或多种性状的转基因植物中的应用;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。
本发明还提供了一种培育具有营养生长期长、叶片品质好和抗倒伏能力强中的一种或多种性状的转基因植物的方法,包括将拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体***目标植物的基因组中并表达;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。
有益效果:
本发明提供了拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在调控植物营养生长中的应用;所述调控植物营养生长包括延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力中的一种或多种;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。本发明所述拟南芥AT5G10290基因在延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力具有较好的生物学功能;由说明书实施例可知,在培育90天时,表达AT5G10290基因的油菜相对于野生型油菜,其叶片厚度提高了60%,叶片长度提高了21%,叶片长度提高了22.1%,叶片数提高了10%(在培育100天时叶片数提高了42.9%~100%),说明表达AT5G10290基因的植物可以提高植物叶片品质;在培育100天时,野生型油菜达到开花期,而表达AT5G10290基因的油菜未开花,说明表达AT5G10290基因的植物可以延长开花期,从而延长植物营养生长期;在培育100天时,表达AT5G10290基因的油菜相对于野生型油菜,其株高降低了52.8%~54%,说明表达AT5G10290基因的植物可以提高植物抗倒伏能力。
附图说明
图1为14株油菜AT5G10290基因全长CDS扩增电泳图;其中LOE-1~LOE-14分别代表筛选的14株油菜;WT为阴性对照;
图2为AT5G10290基因的亚细胞定位结果;
图3为野生型油菜和过表达株系的营养生长时期俯视对比图,其中过表株系1为实施例3筛选得到的过表达-1油菜;
图4为野生型油菜和过表达株系的营养生长时期主视对比图;
图5为野生型油菜和过表达株系的开花时期对比图。
具体实施方式
本发明提供了拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在调控植物营养生长中的应用;所述调控植物营养生长包括延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力中的一种或多种;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示:MRMFSLQKMAMAFTLLFFACLCSFVSPDAQGDALFALRISLRALPNQLSDWNQNQVNPCTWSQVICDDKNFVTSLTLSDMNFSGTLSSRVGILENLKTLTLKGNGITGEIPEDFGNLTSLTSLDLEDNQLTGRIPSTIGNLKKLQFLTLSRNKLNGTIPESLTGLPNLLNLLLDSNSLSGQIPQSLFEIPKYNFTSNNLNCGGRQPHPCVSAVAHSGDSSKPKTGIIAGVVAGVTVVLFGILLFLFCKDRHKGYRRDVFVDVAGEVDRRIAFGQLKRFAWRELQLATDNFSEKNVLGQGGFGKVYKGVLPDNTKVAVKRLTDFESPGGDAAFQREVEMISVAVHRNLLRLIGFCTTQTERLLVYPFMQNLSLAHRLREIKAGDPVLDWETRKRIALGAARGFEYLHEHCNPKIIHRDVKAANVLLDEDFEAVVGDFGLAKLVDVRRTNVTTQVRGTMGHIAPEYLSTGKSSERTDVFGYGIMLLELVTGQRAIDFSRLEEEDDVLLLDHVKKLEREKRLGAIVDKNLDGEYIKEEVEMMIQVALLCTQGSPEDRPVMSEVVRMLEGEGLAERWEEWQNVEVTRRHEFERLQRRFDWGEDSMHNQDAIELSGGR。本发明所述拟南芥AT5G10290基因属于leucine-rich repeat transmembraneprotein kinase family protein(富含亮氨酸重复跨膜蛋白激酶家族蛋白),在GenBank中的编号是AT5G10290,该基因的CDS长1842bp,核苷酸序列优选如SEQ ID No.2所示,编码613个氨基酸,氨基酸序列如SEQ ID No.1所示。
在本发明中,所述植物优选包括十字花科植物,更优选包括油菜或拟南芥。本发明优选通过提高叶片厚度和面积用以提高植物叶片品质,并通过降低植物株高以提高植物抗倒伏能力。
在本发明中,所述拟南芥AT5G10290基因的核苷酸序列优选如SEQ ID No.2所示:ATGAGAATGTTCAGCTTGCAGAAGATGGCTATGGCTTTTACTCTCTTGTTTTTTGCCTGTTTATGCTCATTTGTGTCTCCAGATGCTCAAGGGGATGCACTGTTTGCGTTGAGGATCTCCTTACGTGCATTACCGAATCAGCTAAGTGACTGGAATCAGAACCAAGTTAATCCTTGCACTTGGTCCCAAGTTATTTGTGATGACAAAAACTTTGTCACTTCTCTTACATTGTCAGATATGAACTTCTCGGGAACCTTGTCTTCAAGAGTAGGAATCCTAGAAAATCTCAAGACTCTTACTTTAAAGGGAAATGGAATTACGGGTGAAATACCAGAAGACTTTGGAAATCTGACTAGCTTGACTAGTTTGGATTTGGAGGACAATCAGCTAACTGGTCGTATACCATCCACTATCGGTAATCTCAAGAAACTTCAGTTCTTGACCTTGAGTAGGAACAAACTTAATGGGACTATTCCGGAGTCACTCACTGGTCTTCCAAACCTGTTAAACCTGCTGCTTGATTCCAATAGTCTCAGTGGTCAGATTCCTCAAAGTCTGTTTGAGATCCCAAAATATAATTTCACGTCAAACAACTTGAATTGTGGCGGTCGTCAACCTCACCCTTGTGTATCCGCGGTTGCCCATTCAGGTGATTCAAGCAAGCCTAAAACTGGCATTATTGCTGGAGTTGTTGCTGGAGTTACAGTTGTTCTCTTTGGAATCTTGTTGTTTCTGTTCTGCAAGGATAGGCATAAAGGATATAGACGTGATGTGTTTGTGGATGTTGCAGGTGAAGTGGACAGGAGAATTGCATTTGGACAGTTGAAAAGGTTTGCATGGAGAGAGCTCCAGTTAGCGACAGATAACTTCAGCGAAAAGAATGTACTTGGTCAAGGAGGCTTTGGGAAAGTTTACAAAGGAGTGCTTCCGGATAACACCAAAGTTGCTGTGAAGAGATTGACGGATTTCGAAAGTCCTGGTGGAGATGCTGCTTTCCAAAGGGAAGTAGAGATGATAAGTGTAGCTGTTCATAGGAATCTACTCCGTCTTATCGGGTTCTGCACCACACAAACAGAACGCCTTTTGGTTTATCCCTTCATGCAGAATCTAAGTCTTGCACATCGTCTGAGAGAGATCAAAGCAGGCGACCCGGTTCTAGATTGGGAGACGAGGAAACGGATTGCCTTAGGAGCAGCGCGTGGTTTTGAGTATCTTCATGAACATTGCAATCCGAAGATCATACATCGTGATGTGAAAGCAGCTAATGTGTTACTAGATGAAGATTTTGAAGCAGTGGTTGGTGATTTTGGTTTAGCCAAGCTAGTAGATGTTAGAAGGACTAATGTGACTACTCAAGTTCGAGGAACAATGGGTCACATTGCACCAGAATATTTATCAACAGGGAAATCATCAGAGAGAACCGATGTTTTCGGGTATGGAATTATGCTTCTTGAGCTTGTTACAGGACAACGCGCAATAGACTTTTCACGTTTGGAGGAAGAAGATGATGTCTTGTTACTTGACCACGTGAAGAAACTGGAAAGAGAGAAGAGATTAGGAGCAATCGTAGATAAGAATTTGGATGGAGAGTATATAAAAGAAGAAGTAGAGATGATGATACAAGTGGCTTTGCTTTGTACACAAGGTTCACCAGAAGACCGACCAGTGATGTCTGAAGTTGTGAGGATGTTAGAAGGAGAAGGGCTTGCGGAGAGATGGGAAGAGTGGCAAAACGTGGAAGTCACGAGACGTCATGAGTTTGAACGGTTGCAGAGGAGATTTGATTGGGGTGAAGATTCTATGCATAACCAAGATGCCATTGAATTATCTGGTGGAAGATGA。本发明对所述拟南芥AT5G10290基因的来源没有特殊限制,采用本领域所熟知的拟南芥AT5G10290基因的来源即可。在本发明实施例中,所述拟南芥AT5G10290基因委托基因合成公司合成。
在本发明中,所述包含拟南芥AT5G10290基因的表达载体的基础载体优选包括pEGAD;所述包含拟南芥AT5G10290基因的表达载体的构建方法,优选包括将所述拟南芥AT5G10290基因***pEGAD的EcoRI/SmaI多克隆位点处;具体的构建方法优选参见中国专利CN 110592137 B,在此不再赘述。
本发明还提供了拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在培育具有营养生长期长、叶片品质好和抗倒伏能力强中的一种或多种性状的转基因植物中的应用;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。在本发明中,所述植物优选包括十字花科植物,更优选包括油菜或拟南芥。
本发明还提供了一种培育具有营养生长期长、叶片品质好和抗倒伏能力强中的一种或多种性状的转基因植物的方法,包括将拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体***目标植物的基因组中并表达;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示。在本发明中,所述植物优选包括十字花科植物;更优选包括油菜或拟南芥。本发明验证了所述拟南芥AT5G10290基因在延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力具有较好的生物学功能,将将拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体***目标植物的基因组中并表达可以培育具有营养生长期长、叶片品质好和抗倒伏能力强中的一种或多种性状的转基因植物。
为了进一步说明本发明,下面结合实施例对本发明提供的拟南芥AT5G10290基因在调控植物生长中的应用进行详细地描述,但不能将它们理解为对本发明保护范围的限定。
实施例1
AT5G10290基因过表达载体(35S::AT5G10290)的构建方法,具体构建方法参见中国专利CN 110592137 B中说明书实施例1。
实施例2
AT5G10290基因过表达载体的农杆菌转化及鉴定,具体构建方法参见中国专利CN110592137 B中说明书实施例2。
实施例3
以Westar野生型油菜的幼苗作为转基因受体材料,用实施例2中鉴定为正确转化了表达载体的农杆菌侵染受体材料,将带有目的基因的载体***到受体材料的基因组中,收获种子(具体方法参见中国专利CN 110592137 B中说明书实施例3,区别在于,将步骤(6)中被侵染拟南芥替换为Westar野生型油菜的愈伤组织,侵染后的愈伤组织继续在分化培养基上进行分化培养;所述分化培养基以MS培养基为基础培养基,还含有30g/L蔗糖和3g/LPhytagel,所述分化培养基的pH值为5.8;培养条件为恒温25℃暗培养;经过1个月的培养,将分化出叶片和根的幼苗移栽到土里继续培养,并得到T1代种子)。
在获得T1代大量转基因种子后,根据表达载体上所携带的卡那霉素抗性基因,将种子播种于加入卡那霉素的培养基上,约2周后初步筛选对除草剂有抗性的转化植株,并移栽到土壤里生长。
经4~5周植物长大后,提取叶片DNA结合PCR扩增目的片段的方法(具体方法参见中国专利CN 110592137 B中说明书实施例7中的2、T-DNA***点的鉴定)进一步鉴定转化植株,一共获得带有全长AT5G10290 CDS序列的油菜14株(见图1),但是经过激光共聚焦显微镜观测该基因融合表达的EGFP信号后(具体方法参见中国专利CN 110592137 B中说明书实施例5),只有两个株系检测到了荧光信号,分别将它们标记为过表达1和过表达8(见图2,其中Auto-fluo表示的是叶绿体的自发荧光,而非目的荧光信号;Merge为把EGFP和Auto-fluo图层叠加,目的是为表明EGFP信号为目的蛋白特异性荧光信号,而非细胞自发的非特异性荧光信号)。
经鉴定的植株单株收种子,之后再繁种一代重复以上筛选方法。直至所有转化植株都具有显著抗性且证明含有目的片段后,对其提取RNA做基因表达检测。用T3或T4代的转基因植株(过表达-1和过表达-8)进行后续试验。
实施例4
将野生型油菜、过表达-1油菜和过表达-8油菜分别播种在直径30厘米,高40厘米的塑料圆盆中,在室外自然条件下培养,每周充分浇水一次,统计培育90天时不同株系的叶片厚度、叶片长度、叶片宽度和叶片数,统计结果见表1(对照图见图3和图4);统计培育90天时不同株系的叶片数、花葶高度、开花数量和株高,统计结果见表2(对照图见图5)。
表1培育90天时株系的营养生长期比较(第7~8片叶)
野生型 | 过表达-1 | 过表达-8 | |
叶片厚度(mm) | 0.5±0.1 | 0.8±0.2 | 0.7±0.1 |
叶片长度(cm) | 13.8±0.5 | 16.7±0.3 | 17.0±0.4 |
叶片宽度(cm) | 9.5±1.0 | 11.6±1.0 | 10.9±1.1 |
叶片数(片) | 10.0±1.0 | 11.0±1.0 | 11.0±1.2 |
表2培育110天时株系开花时期对比
野生型 | 过表达-1 | 过表达-8 | |
叶片数(片) | 7±2 | 14±3 | 10±2 |
花葶高度(cm) | 25±5 | 0±0 | 0±0 |
开花数量(朵) | 14±3 | 0±0 | 0±0 |
株高(cm) | 125±11 | 59±7 | 57±5 |
发现过表达株系的叶片变厚、变大(见图3和图4)。而且野生型植株的开花期显著早于过表达株系(图3)
由图3~图5可知,表达株系相对于野生型株系的叶片明显变厚、变大、叶片数增加和株高明显降低;而且野生型植株的开花期显著早于过表达株系。
由表1和表2可知,在培育90天时,表达AT5G10290基因的油菜相对于野生型油菜,其叶片厚度提高了60%,叶片长度提高了21%,叶片长度提高了22.1%,叶片数提高了10%(在培育100天时叶片数提高了42.9%~100%),说明表达AT5G10290基因的植物可以提高植物叶片品质;在培育100天时,野生型油菜达到开花期,而表达AT5G10290基因的油菜未开花,说明表达AT5G10290基因的植物可以延长开花期,从而延长植物营养生长期;在培育100天时,表达AT5G10290基因的油菜相对于野生型油菜,其株高降低了52.8%~54%,说明表达AT5G10290基因的植物可以提高植物抗倒伏能力。
综上所述,拟南芥AT5G10290基因在延长植物营养生长期、提高植物叶片品质和提高植物抗倒伏能力具有较好的生物学功能。
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 中国科学院昆明植物研究所
<120> 拟南芥AT5G10290基因在调控植物生长中的应用
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gtcacgagac gtcatgagtt tgaacggttg cagaggagat ttgattgggg tgaagattct 1800
atgcataacc aagatgccat tgaattatct ggtggaagat ga 1842
Claims (6)
1.拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在调控植物营养生长中的应用,其特征在于,所述调控植物营养生长为提高植物抗倒伏能力;所述提高植物抗倒伏能力为降低植物株高;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ IDNo.1所示;所述植物为十字花科植物;所述十字花科植物为油菜。
2.根据权利要求1所述的应用,其特征在于,所述拟南芥AT5G10290基因的核苷酸序列如SEQ ID No.2所示。
3.根据权利要求1或2所述的应用,其特征在于,所述包含拟南芥AT5G10290基因的表达载体的基础载体包括pEGAD。
4.根据权利要求3所述的应用,其特征在于,所述包含拟南芥AT5G10290基因的表达载体的构建方法,包括将所述拟南芥AT5G10290基因***pEGAD的EcoRI/SmaI多克隆位点处。
5.拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体在培育抗倒伏能力强的转基因植物中的应用;所述抗倒伏能力强为株高低于野生型;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQ ID No.1所示;所述植物为十字花科植物;所述十字花科植物为油菜。
6.一种培育抗倒伏能力强的转基因植物的方法,其特征在于,包括将拟南芥AT5G10290基因或包含拟南芥AT5G10290基因的表达载体***目标植物的基因组中并表达;所述抗倒伏能力强为株高低于野生型;所述拟南芥AT5G10290基因的编码蛋白的氨基酸序列如SEQID No.1所示;所述植物为十字花科植物;所述十字花科植物为油菜。
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