CN103351429A - 调控植物油脂与脂肪酸的转录因子及其编码基因与应用 - Google Patents
调控植物油脂与脂肪酸的转录因子及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一个调控植物油脂与脂肪酸的转录因子及其编码基因与应用。本发明所提供的调控植物油脂和脂肪酸的转录因子,命名为VfLEC1,其氨基酸序列如SEQIDNO:1所示。本发明的转录因子及其编码基因对于植物油脂与脂肪酸的成分和含量起到调控作用。
Description
技术领域
本发明属于植物基因工程领域,具体的说,本发明涉及一个调控植物油脂与脂肪酸的转录因子及其编码基因与应用。
背景技术
植物油脂作为主要食用油和生物质能源而备受关注。大多数植物中,植物油脂以三酰甘油(Triacylglycerol,TAG)的形式贮藏于植物种子中,富含不饱和脂肪酸。
植物油脂的生物合成是一个复杂的生理生化过程,主要包括脂肪酸及脂酰辅酶A的生成、3-磷酸甘油与游离脂肪酸脱水缩合3个过程。脂肪酸合成主要在质体中进行。在种子的发育过程中,光合作用产生的蔗糖为合成脂肪酸的主要碳源,通过糖酵解途径生成己糖,并进一步生成丙酮酸。在丙酮酸羧化酶催化下生成脂肪酸合成的前体乙酰辅酶A(acetyl-CoA)。随后,乙酰辅酶A在乙酰辅酶A羧化酶(acetyl-CoA carboxylase, ACCase)的催化下,形成丙二酰单酰辅酶A,后者在脂肪酸合成酶的作用下进行连续聚合反应,每次增加两个碳,酰基碳链又与酰基载体蛋白(acyl carrier protein, ACP)结合进入脂肪酸合成途径。随后的脂肪酸修饰和三酰甘油酯的合成等主要在内质网上进行。酰基辅酶A在内置网上经3-磷酸甘油酰基转移酶(GPAT)、溶血性磷脂酸酰基转移酶(LPAAT)、二酰甘油转酰酶(DGAT)以及磷脂酸磷酸水解酶(PAPase)的作用,分别在甘油骨架上附连脂肪酸合成三酰甘油酯。科学家们通过对上述关键酶基因的调控以提高油脂含量和改良脂肪酸成分进行了有益的探索并取得了一定的进展。Roesaler等利用种子特异性启动子在油菜中过表达 ACCase基因,使其种子的含油量提高3-5%(Roesler et al., 1997, Plant physiology. 113: 75-81)。同时,通过研究发现,植物体内脂肪酸和油脂合成是一个复杂的网络调控,对个别或单个基因的调控还不能有效地达到改变油脂含量和优化脂肪酸成分的目的。因此,通过对转录因子或蛋白激酶的调控,可以达到调控多个基因及网络的目的(Girke et al., Plant Physiol. 2000,124(4):1570-81.)。
通过对模式植物和作物的研究,发现转录因子对脂肪酸的重要调控作用,其中LEC(Leafy Cotyledon 1)是参与脂肪酸代谢最重要的转录因子之一。LEC编码CCAAT-box结合因子HAP3(Heme-activated protein 3)亚基的同源物。过量表达LEC1可以使主要脂肪酸组分含量大幅度提高(Tan et al., Plant Physiol. 2011, 156(3):1577-88. Mu et al., Plant Physiol. 2008,148 (2):1042-54)。而木本油料植物中调控油脂和脂肪酸的关键因子相关的文献报道较少。然而,对一些化工油料植物特殊油脂和脂肪酸的分子解析,可以更加丰富植物油脂的基因资源。申请人在在特殊工业用途木本油料植物油桐的转录组中也发现了约占2.5%的转录因子(Chen, et al., Industrial Crops and Products 2010,32: 684–686),并对其在油脂含量与成分合成中的调控作用进行了积极的探索。
发明内容
本发明的目的是提供一个调控植物油脂和脂肪酸的转录因子。
本发明所提供的调控植物油脂和脂肪酸的转录因子,命名为VfLEC1,来源于油桐(Vernicia fordii),是如下1)或2)的蛋白:
1) 由序列表中序列1所示的氨基酸序列组成的蛋白质;
2) 在序列表中序列1的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且与植物脂肪酸代谢相关的由1)衍生的蛋白质。
其中,序列表中的序列1由242个氨基酸残基组成。其中,自氨基端第71-136位氨基酸为保守区域。
所取代和/或缺失和/或添加一个或几个氨基酸残基,可以是非结构域中的氨基酸残基,其改变不会对蛋白的功能产生影响。
上述是2)中VfLEC1可人工合成,也可先合成其编码基因,再进行生物表达得到。上述2)中的VfLEC1的编码基因可通过将序列表中序列2自5’端第33-796位碱基所示的DNA序列中确实一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5’端和/或3’端连上标签编码序列得到。
上述以种子脂肪酸合成相关蛋白的cDNA基因(命名为VfLEC)也属于本发明的保护范围。
与种子脂肪酸合成相关的cDNA基因具体可为如下1)-4)中任何一所述的基因:
1) 其编码序列是序列表中序列2的自5’末端第33-796位脱氧核糖核苷酸;
2) 其核苷酸序列是序列表中的序列2;
3) 在严格条件下与1)或2)的基因杂交且编码所述蛋白的基因;
4) 与1)或2)的基因具有90%以上同源性且编码所述蛋白的基因。
序列表中的序列2由1334个碱基组成,其开放阅读框架(Open Reading Frame,ORF)为自5’末端第33-796位碱基,编码序列表中序列1的氨基酸序列VfLEC1蛋白;序列2自5’末端第71-136位碱基编码NF-YB结构域。
所述编码调控植物脂肪酸合成转录因子的基因既可以VfLEC1的cDNA序列,也可畏VfLEC1的基因组基因序列,与VfLEC1具有90%以上同源性且编码相同蛋白的DNA序列,是将VfLEC1的cDNA用已知的方法进行分离和/或修饰和/或设计得到的。
所述编码调控植物脂肪酸合成转录因子的基因VfLEC1或其同源序列可以正义方向或反义方向导入植物组织、细胞或器官。
扩增上述VfLEC1基因全长或其任一片段的引物也属于本发明的保护范围。
含有上述与种子脂肪酸合成先关的蛋白编码基因的重组载体、转基因细胞系和重组菌也属于本发明的保护范围。
附图说明
图1为VfLEC1与已知其它物种同源基因的进化关系树。
图2为重组pB110- VfLEC1载体。pB110- VfLEC1含有植物种子表达特异性启动子β-半大豆球蛋白(Beta-Conglycinin)启动子,位于***基因VfLEC1基因序列之前。
图3为VfLEC1转基因拟南芥种子中油脂含量成分变化。
图4为VfLEC1转基因拟南芥种子中脂肪酸成分含量变化。
具体实施方式
本发明的以下实施例所使用的分子生物学和生物化学方法均为已知的技术。在Current Protocols in Molecular Biology(Ausubel编写,John Wiley and Sons公司出版),和J. Sambrook等编写Cold Spring Harbor Laboratory Press (2001) 出版的Molecular Cloning: A Labortory Manual, 3rd ED.等文献均有详细的说明。
实施例1、与植物脂肪酸和油脂合成相关的基因VfLEC1的分离
(1)总RNA的提取及mRNA的纯化
在用Trizol方法提取总RNA时增加了氯仿抽提次数,得到的总RNA利用1%琼脂糖凝胶电泳可检测到28S RNA、18S RNA和5S RNA三条带,且28S RNA与18S RNA的亮度比例约为2:1,5S RNA条带较弱,说明总RNA没有降解,较完整,完全符合构建高质量cDNA文库的要求。用分光光度计检测OD260/280比值在1.9-2.0之间。在用磁珠法分离纯化mRNA时,将总RNA浓缩后再过磁珠得到的mRNA质量较好。
(2) ds cDNA合成及分级分离
由于按照Stratagene公司的ZAP-cDNA Gigapack III Gold Cloning Kit 使用说明,在分级分离时,需要进行装柱,时间比较长,且容易产生气泡,故改选用Clontech公司的Creator? SMART? cDNA Library Construction Kit中分级分离部分的 CHROMA SPIN-400 Column,收集片段长度在500 bp以上的过柱液并混合到同一管中。
(3) cDNA文库质量的检测
按照Stratagene 公司的ZAP-cDNA Gigapack III Gold Cloning Kit检测,油桐种仁 cDNA 初级文库的滴度为1×106 Pfu/mL,重组率为 99.7%,扩增后文库的滴度为1.2×109 Pfu/mL,随机挑选20个阳性克隆进行 PCR 检测,琼脂糖凝胶电泳检测结果表明***片段大小在 0.5~2.5 kb 之间,平均约1 kb,对环化后质粒DNA进行EcoR I 和Xho I 双酶切,检测结果与 PCR 一致。文库的滴度、重组率及完整性均符合构建高质量文库标准。
(4)油桐种仁cDNA初始文库送上海生物芯片公司进行测序,获得转录因子VfLEC序列。测得的原始序列利用crossmatch软件去除5’端载体序列、文库接头序列和poly(A)序列;用phrap进行拼接;通过本地BLAST获得515个与其他序列无同源区的序列(singlets),其余的1849条序列依照同源性聚为228个contigs。转录因子VfLEC序列通过重叠群拼接获得。
(5)VfLEC基因的分离
格局VfLEC的序列设计特异性引物从油桐cDNA文库中扩增,获得特异片段后,进行TA克隆测序。
实施例2、转VfLEC基因拟南芥的获得
为更好地验证VfLEC 的功能,选择野生型拟南芥(Col-0)、突变体拟南芥。突变体拟南芥购自 Arabidopsis Biological Resource Center (ABRC; Ohio State University) ,分别是拟南芥LEC1基因(GenBank 登录号AT1G21970)的不同部位的突变体,商品号为SALK_000450。
构建重组植物表达载体pB110-VfLEC,电激转化农杆菌GV3101,通过卡纳抗生素筛选阳性转化子。农杆菌介导法转化拟南芥,通过红色荧光筛选转基因种子。
实施例3、转VfLEC基因拟南芥种子油脂和脂肪酸检测
收集野生型拟南芥和转基因拟南芥的种子,60℃烘干过夜,粉碎机磨碎再烘干,索氏抽提法提取油脂,差重法计算含油量,GC-MS法检测脂肪酸成分变化。
如图3、4结果显示,转VfLEC基因的拟南芥种子内油脂含量提升5.6%-7.1%,脂肪酸含量大幅度提升,且VfLEC转突变体拟南芥中油脂含量、油酸和亚油酸含量高于野生型,这说明,VfLEC基因可以补偿拟南芥LEC1的功能,并在一定程度上优于拟南芥LEC1基因。
序列表
Sequence ID 1
<110> 中国林科院亚热带林业研究所
<120> 油桐调控油脂合成转录因子编码基因vfLEC
<130> patent
<160> 3
<170> PatentIn version 3.2
<210> 1
<211> Length : 242
SequenceName : vfLEC
<212> Type : DNA
<221> CDS
<213> 油桐(Valernicia fordii)
<400> 1
Met Glu Arg Gly Gly Arg Phe His Arg Tyr Arg Arg His Ala Lys
1 5 10 15
Gln Gln Ala Ser Ile Ser Ser Ala Ala Ser Ser Gly Val Asn Leu
20 25 30
Leu Arg Ala Asn Asn Pro Pro Asn Phe Asn Leu Pro Thr Asn Leu
35 40 45
Asn Pro Ser Gln Ala Asn Pro Thr Met Val Pro Asn Pro Gln Gln
50 55 60
Pro Pro Gln Cys Thr Val Arg Glu Gln Asp Gln Tyr Met Pro Ile
65 70 75
Ala Asn Val Ile Arg Ile Met Arg Arg Ile Leu Pro Ala His Ala
80 85 90
Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys Val Ser
95 100 105
Glu Tyr Ile Ser Phe Ile Thr Gly Glu Ala Asn Asp Arg Cys Gln
110 115 120
His Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp Val Leu Trp Ser
125 130 135
Met Gly Lys Leu Gly Phe Asp Asp Tyr Val Glu Pro Leu Thr Leu
140 145 150
Phe Leu Asn Arg Tyr Arg Glu Ala Glu Asn Glu Arg Gly Ser Val
155 160 165
Arg Asp Pro Leu Leu Lys Arg Ser Asn Val Gly Val Val Asp Tyr
170 175 180
Gly Asn Ile Gly Met Pro Pro Phe Met Pro Ser Phe Ala Val Gly
185 190 195
Pro Pro Pro Pro Gln Gly Val Phe Asp Pro Ala Met Phe Gly Ala
200 205 210
Tyr Tyr Arg Asp Val Ser Asp Ala Gly Ala Gly Met Gly Gly Pro
215 220 225
Ser Ser Ser Asn Asn Pro Tyr Ile Asn Phe Asp Pro Phe Thr Gln
230 235 240
Phe Lys
SEG ID NO:2
<210> 2
<211> 1334
<212> DNA
<213> 油桐(Vernicia fordii)
<400> 2
cattttattt ttattattat tttagtttaa ttatggaacg tggaggcagg ttccatcgct 60
atcgaaggca tgcaaaacag caagcttcaa taagctctgc tgctagctct ggcgtgaatt 120
tactgcgagc aaataaccca ccaaacttca atctccccac caatctgaac ccaagtcagg 180
ccaacccaac aatggtgcca aacccccagc agccacctca atgcactgtg cgtgaacaag 240
atcagtacat gccaatagcc aatgtgattc gcatcatgcg ccgtattctc ccagctcatg 300
caaaaatctc tgatgatgcc aaagaaacta tccaagaatg tgtctctgaa tacataagct 360
tcatcactgg agaggcaaat gatcgttgcc agcatgagca gcgcaagact atcactgctg 420
aggatgtgct ttggtctatg ggaaagctgg gattcgatga ctatgtcgag cctctaactc 480
tttttctgaa tcgctatcgc gaggcagaaa atgaacgtgg ctctgttcgt gatccactcc 540
tgaagcgtag caatgttggt gttgttgatt atggaaatat tgggatgcct ccttttatgc 600
caagttttgc tgtgggacct cccccaccac agggtgtttt tgatcctgca atgtttggtg 660
cctattatag ggatgtttct gatgctggtg ctggtatggg tggcccctct tcttcgaaca 720
atccttatat caacttcgat ccatttactc agttcaagtg atgacagttt gatagttcga 780
caagaaagga ggaacatata tgactattaa ctataaaaga aataaaggga taatatcata 840
aacttttttt tttttaaatt aaagaaatgt agtttaagga gtagatggca ctagtgacta 900
gctagtgaga taccatttgc tcctcctcct cctcctttta tatgttgttt acttctatct 960
gatgtaacaa catacctgct atattttgaa tttcaagcat atatatggaa agaaatttca 1020
attaaaaaaa aaaaaaaaaa actcgagggg gggcccggta cccaattcgc cctatagtga 1080
gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 1140
tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 1200
ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcaaattgt 1260
aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 1320
ccaataggcc gaaa 1380
Claims (9)
1.一个调控植物油脂与脂肪酸的转录因子,其氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述转录因子的cDNA基因序列。
3.根据权利要求2所述的cDNA基因序列,其特征在于,该cDNA序列的碱基序列如SEQ ID NO:2所示。
4.含有权利2或3所述基因的表达载体。
5.含有权利2或3所述基因的转基因细胞系。
6.含有权利2或3所述基因的宿主菌。
7.一种调控植物油脂与脂肪酸组分和含量的方法,其特征在于,是将权利2或3所述的基因导入植物组织、细胞或器官,植物脂肪酸组分和含量得到调控。
8.根据权利要求7所述的方法,其特征在于,所述基因通过含有该基因的植物表达载体导入植物组织、细胞或器官;用于构建所述植物表达载体的出发载体为植物表达载体。
9.根据权利要求7或8所述的方法,其特征在于,所述植物为双子叶植物。
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