CN114426972A - 花生转录调控因子AhSAP1基因在调控种子大小中的应用 - Google Patents

花生转录调控因子AhSAP1基因在调控种子大小中的应用 Download PDF

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CN114426972A
CN114426972A CN202210146687.4A CN202210146687A CN114426972A CN 114426972 A CN114426972 A CN 114426972A CN 202210146687 A CN202210146687 A CN 202210146687A CN 114426972 A CN114426972 A CN 114426972A
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庄伟建
陈华
谢文萍
张冲
杨强
蔡铁城
党浩
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Abstract

本发明属于生物技术领域，公开了花生转录调控因子AhSAP1基因在调控种子大小中的应用。AhSAP1基因核苷酸序列如SEQ ID No.1所示。通过Gateway系统构建CaMV 35S启动子驱动的过量表达载体经农杆菌介导转化拟南芥，建立了转基因拟南芥株系。将转基因拟南芥株系与野生型植株比较分析发现，AhSAP1在拟南芥中超量表达可显著增加叶片、花器官、角果及种子大小。本发明为利用基因工程手段培育大果植物新品种提供了重要的基因资源，可用于提高作物的产量，具有重要的应用前景。

Description

花生转录调控因子AhSAP1基因在调控种子大小中的应用
技术领域
本发明属于植物基因工程技术领域，具体涉及花生转录调控因子AhSAP1基因在调控种子大小中的应用。
背景技术
种子大小是重要的发育生物学问题，是决定作物产量的关键因素，长期以来一直都是作物遗传改良的主要目标之一。解析调控种子大小的分子机制，能为作物的高产育种提供重要的理论基础和基因资源。
已知泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）主要由泛素、泛素活化酶、泛素结合酶、泛素连接酶和26S蛋白酶体组成，是细胞内重要的蛋白质降解途径，通过影响蛋白质的稳定性、活性和定位来参与多种细胞代谢过程。越来越多研究表明，泛素-蛋白酶体途径在调控种子大小过程中具有重要作用。拟南芥PPD1和PPD2基因可通过促进分生组织细胞增殖调控器官大小。PPD与KIX蛋白形成转录抑制复合体KIX-PPD来调控植物叶片大小，STERILE APETALA (SAP)/SUPPRESSOR OF DA1 (SOD3) 是F-box蛋白，参与构成SKP1/Cullin/F-box（SCF）E3泛素连接酶复合物，通过促进分生组织细胞的增殖来控制器官大小。SAP能够降解PPD，调控KIX-PPD复合体蛋白稳定性。KIX8/9是SAP的底物，KIX8/9和PPD1/2突变后，引起外皮中的细胞增殖和细胞伸长增加，导致种子变大。KIX8/9和PPD1/2与转录因子MYC3/4相互作用，形成KIX-PPD-MYC复合体。该复合体可与GIF1启动子中的G-box结合，并抑制GIF1基因的表达。遗传分析表明，GIF1在SAP-KIX-PPD-MYC途径的下游发挥作用，调控种子大小。
豆科植物中与拟南芥PPD1和PPD2序列相似度较高的BS1编码植物特异转录因子TIFY，参与调控种子大小。苜蓿bs1-1突变体种子变大、变重。大豆中BS1基因受到抑制后种子变大。苜蓿SLB1为拟南芥SAP的同源基因，通过调控BS1的稳定性控制器官大小，在苜蓿和大豆中过量表达SLB1可使种子变大。以上结果表明，SAP基因在提高豆科作物产量方面具有重要的应用价值。
发明内容
本发明提供一种花生转录调控因子AhSAP1基因及其在调控种子大小中的应用，将为利用基因工程手段培育大果植物新品种提供重要的基因资源，用于提高作物的产量，具有重要的应用前景。
为实现上述目的，本发明采用如下技术方案：
一种调控植物种子大小的花生转录调控因子AhSAP1基因，其CDS序列长度为1455bp，所述基因的核苷酸序列如SEQ ID NO.1所示。
一种花生转录调控因子AhSAP1基因编码的蛋白，所述AhSAP1基因编码484个氨基酸的蛋白，具体氨基酸序列如SEQ ID NO.2 所示。
一种包含上述花生转录调控因子AhSAP1基因的超量表达载。
进一步的，上述超量表达载体的构建方法为：基于Gateway系统通过BP反应构建入门载体pDONR207-AhSAP1，再经过LR反应构建CaMV 35S启动子驱动的植物超表达载体pK7WG2.0-AhSAP1。
上述花生转录调控因子AhSAP1基因在调控植物种子大小中的应用，所述植物包括但不限于拟南芥、花生。
上述花生转录调控因子AhSAP1基因编码的蛋白在调控植物种子大小中的应用。
上述包含上述花生转录调控因子AhSAP1基因的超量表达载在调控植物种子大小中的应用。
本发明基于前期QTL定位、BSA与BSR分析获得一个转录调控因子STERILE APETALA（SAP），命名为AhSAP1。花生转录调控因子AhSAP1基因过量表达能够显著增加叶片、花器官、角果及种子大小，这为花生高产分子育种提供了理论基础。
本发明的有益效果在于：
基于前期QTL定位、BSA与BSR分析获得一个转录调控因子AhSAP1。经过PCR克隆获得编码该蛋白的cDNA序列，通过基于Gateway系统的BP和LR反应构建CaMV 35S启动子驱动AhSAP基因植物表达载体pK7WG2.0-AhSAP1，将其转化GV3101农杆菌，通过浸花法将其导入拟南芥，对转基因拟南芥进行表型鉴定，结果证明AhSAP1可以正向调控种子大小。本发明为利用基因工程手段培育大果植物新品种提供重要的基因资源，用于提高作物的产量，具有重要的应用前景。
附图说明
图1为基于Gateway系统的花生AhSAP1超量表达载体的构建示意图。
图2为花生AhSAP1超量表达（AhSAP1-OE）转基因拟南芥花、角果与种子大小。1：野生型拟南芥中Col-0；2：转空载体对照：3：AhSAP1过量表达。
具体实施方式
实施例1 花生转录调控因子AhSAP1基因的筛选
通过QTL定位、BSA与BSR分析获得一个转录调控因子STERILE APETALA（SAP），命名为AhSAP1。
根据其预测的AhSAP1基因CDS序列设计引物，进行聚合酶联反应扩增该基因，结果表明，AhSAP1基因CDS序列含有1455个碱基对，编码484个氨基酸，其核苷酸序列如SEQ IDNO.1所示，其编码的氨基酸序列如SEQ ID NO.2 所示。
实施例2 构建AhSAP1超量表达载体
根据AhSAP1全长基因CDS序列，设计特异性引物（AhSAP1-attB1-F: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCATCATCACCATCATCATC-3’，AhSAP1-attB2-R: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTC-
CTATACACCAAAATCCAATAAC-3’）以花生粤油92的cDNA为模板进行PCR扩增，采用TAKARA公司高保真酶PrimeSTAR®MAX进行扩增，PCR反应体系：1 µL的cDNA为模板，10 µL的2×PrimeSTAR®MAX mix，正反引物各0.5 µL，补水至20 µL。反应条件：95℃预变性5 min；95℃ 30 s， 60℃ 30 s，72℃ 2 min，28个循环。PCR产物琼脂糖凝胶电泳检测后切胶纯化回收，将目的基因片段与pDONR207空载连接进行BP反应：纯化后的AhSAP1产物1 μL（80~100ng），pDONR207空载体1 μL，BP酶0.25 μL，25℃连接过夜，将连接产物转化到大肠杆菌DH5α感受态细胞，筛选阳性克隆进行测序，测序正确的克隆提取质粒，构建入门载体pDONR207-AhSAP1。将入门载体质粒和植物超量表达载体pK7WG2.0进行LR反应：pDONR207-AhSAP1质粒1 μL（80~100 ng），pK7WG2.0空载1 μL，LR酶0.25 μL，25℃连接过夜，转化大肠杆菌，验证阳性克隆，构建植物超表达载体pK7WG2.0-AhSAP1。pK7WG2.0-AhSAP1超量表达载体的构建示意图如图1所示。
实施例3 超量表达AhSAP1转基因拟南芥表型分析
将实施例1中超表达载体pK7WG2.0-AhSAP1转化农杆菌GV3101，通过浸花法转化至拟南芥，通过卡那霉素筛选和转基因分子鉴定获得阳性转基因株系，共获得了58株AhSAP1-OE转基因阳性拟南芥植株。通过半定量RT-PCR获得提高AhSAP1基因表达水平的转基因T3代株系。将T3代转AhSAP1拟南芥、转空载体拟南芥和野生型拟南芥Col-0种子播种。于花期对花器官进行拍照观察，于角果成熟期对角果及种子进行拍照观察。结果表明，过表达AhSAP1可使花器官变大，角果变粗，种子变大（图2）。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修改，皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福建农林大学
<120> 花生转录调控因子AhSAP1基因在调控种子大小中的应用
<130> 4
<160> 4
<170> PatentIn version 3.3
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<211> 1455
<212> DNA
<213> SEQ ID No.1
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atgtcatcat caccatcatc atcatcatca acatcatcat catcatcatc aacatcttct 60
ccttctcctc ctcctcctcc ttcaaatgct gctgcttccg acgccaatgc tgcccccgac 120
tcttctttcc ggaggtccac ccgcgaattc ggagagtctt ccactgctcg ccagcaacgt 180
ctacgtgcca tgaacgaggc tctccccgag gtccttctcg aaatactcgc tgctcaagtt 240
gccattgatg cttccaaccg ccatggtcgc ttggctgctg ctcctgccgt tgctcgtttg 300
tttcaggttt gttctacatg gcgaggggtc tcacacttgg atccactgtg gcagagactt 360
accagattga tctggcgccg ggcctactta atacgcgaca cgtggcgaga ggagtacatc 420
tactggcatc ggacggctag gaactttgca ttgggagcac cctctttcca tgtcccacac 480
ttcgatcctt ctatgcctcc caatggcgcc ggcgaccgtc aaaccctcct atgccgctgc 540
cttgctctct ctgaccgcca cctcgcctgc ggctttgcgg acggcacggt ccgccttttc 600
gatgttgaga ctctcgccca cgtcagcaca ttccggacta accacggccc gcggttcggc 660
ccgttctccc ggtccgtgtc cggaataatc ctctctaaca gcacgctcac ctttgctaga 720
ctcgacgggg atattttcgt ggcgtcggtc aacgtggcca acgaaccacc gcaacaggtg 780
gttgcaggtg acgtggtgaa caacggtgtt ttagttgagt tcgcgggaaa cagtcggtgg 840
tgggtgggct tatacgcagg ccttccgggc cgggctttcc aaatatggga cgcggccaac 900
cagcaactgg tatcagtggg cgggacgtta acagatccgg aaacggttat gggatggcac 960
atgctaacag aattggttga gccagttggg cgcgtgagag taacggaacc agattacgtc 1020
gtggcgtgca cgagctcgag attagtttgt tttagtttgt ggaacccgga ggaagttata 1080
cgcgacgtgg gttctgttac ggggttcgtt gtaagcagtt tggacgcgcg tcacgatgcg 1140
tttgtgattg tcgagaggaa cggttatggt acggtgcgac gcgccagcac gttcgagcgg 1200
ttgagtaggt tccggttgag gcctccgtgg gttaggggtt tgttagcgtg catcaacttg 1260
ggttatgtgt taacttactc cggggtctct cggatgctaa gggtttggaa cattgagcaa 1320
cttgctgggg ttttctgttg cgcgttggaa gttagggata atgcacctga gggtaatgcc 1380
atggtggcaa atgaaagaca cgtggcgatt tcttggaatg acacctttat acagttattg 1440
gattttggtg tatag 1455
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Met Ser Ser Ser Pro Ser Ser Ser Ser Ser Thr Ser Ser Ser Ser Ser
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Ala Ile Asp Ala Ser Asn Arg His Gly Arg Leu Ala Ala Ala Pro Ala
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Val Ala Arg Leu Phe Gln Val Cys Ser Thr Trp Arg Gly Val Ser His
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Tyr Leu Ile Arg Asp Thr Trp Arg Glu Glu Tyr Ile Tyr Trp His Arg
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Thr Ala Arg Asn Phe Ala Leu Gly Ala Pro Ser Phe His Val Pro His
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Phe Asp Pro Ser Met Pro Pro Asn Gly Ala Gly Asp Arg Gln Thr Leu
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Leu Cys Arg Cys Leu Ala Leu Ser Asp Arg His Leu Ala Cys Gly Phe
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Ala Asp Gly Thr Val Arg Leu Phe Asp Val Glu Thr Leu Ala His Val
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Ser Val Ser Gly Ile Ile Leu Ser Asn Ser Thr Leu Thr Phe Ala Arg
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Leu Asp Gly Asp Ile Phe Val Ala Ser Val Asn Val Ala Asn Glu Pro
245 250 255
Pro Gln Gln Val Val Ala Gly Asp Val Val Asn Asn Gly Val Leu Val
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Glu Phe Ala Gly Asn Ser Arg Trp Trp Val Gly Leu Tyr Ala Gly Leu
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Pro Gly Arg Ala Phe Gln Ile Trp Asp Ala Ala Asn Gln Gln Leu Val
290 295 300
Ser Val Gly Gly Thr Leu Thr Asp Pro Glu Thr Val Met Gly Trp His
305 310 315 320
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325 330 335
Pro Asp Tyr Val Val Ala Cys Thr Ser Ser Arg Leu Val Cys Phe Ser
340 345 350
Leu Trp Asn Pro Glu Glu Val Ile Arg Asp Val Gly Ser Val Thr Gly
355 360 365
Phe Val Val Ser Ser Leu Asp Ala Arg His Asp Ala Phe Val Ile Val
370 375 380
Glu Arg Asn Gly Tyr Gly Thr Val Arg Arg Ala Ser Thr Phe Glu Arg
385 390 395 400
Leu Ser Arg Phe Arg Leu Arg Pro Pro Trp Val Arg Gly Leu Leu Ala
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Cys Ile Asn Leu Gly Tyr Val Leu Thr Tyr Ser Gly Val Ser Arg Met
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Leu Arg Val Trp Asn Ile Glu Gln Leu Ala Gly Val Phe Cys Cys Ala
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Leu Glu Val Arg Asp Asn Ala Pro Glu Gly Asn Ala Met Val Ala Asn
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<212> DNA
<213> AhSAP1-attB1-F
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ggggacaagt ttgtacaaaa aagcaggctt catgtcatca tcaccatcat catc 54
<210> 4
<211> 52
<212> DNA
<213> AhSAP1-attB2-R
<400> 4
ggggaccact ttgtacaaga aagctgggtc ctatacacca aaatccaata ac 52

Claims (8)

1.一种调控植物种子大小的花生转录调控因子AhSAP1基因，其特征在于，所述AhSAP基因的CDS序列长度为1455 bp，其核苷酸序列如SEQ ID No.1所示列。
2.一种调控植物种子大小的花生转录调控因子AhSAP1基因编码的蛋白，其特征在于：所述AhSAP1基因编码484个氨基酸的蛋白，具体氨基酸序列如SEQ ID NO.2所示。
3.一种包含如权利要求1所述一种调控植物种子大小的花生转录调控因子AhSAP1基因的超量表达载体。
4.根据权利要求3所述的一种调控植物种子大小的花生转录调控因子AhSAP1基因的超量表达载体，其特征在于：所述超量表达载体的构建方法包括：基于Gateway系统通过BP反应构建入门载体pDONR207-AhSAP1，再经过LR反应构建CaMV 35S启动子驱动的植物表达载体pK7WG2.0-AhSAP1。
5.如权利要求1所述的花生转录调控因子AhSAP1基因在调控植物种子大小中的应用。
6.根据权利要求5所述的花生转录调控因子AhSAP1基因在调控植物种子大小中的应用，其特征在于：所述植物包括但不限于拟南芥、花生。
7.如权利要求2所述的花生转录调控因子AhSAP1基因编码的蛋白在调控植物种子大小中的应用。
8.如权利要求3所述包含上述花生转录调控因子AhSAP1基因的超量表达载在调控植物种子大小中的应用。
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