CN114703208A - 马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途 - Google Patents

马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途 Download PDF

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CN114703208A
CN114703208A CN202210071575.7A CN202210071575A CN114703208A CN 114703208 A CN114703208 A CN 114703208A CN 202210071575 A CN202210071575 A CN 202210071575A CN 114703208 A CN114703208 A CN 114703208A
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potato
stgapc
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starch content
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何天久
刘腾飞
吴巧玉
夏军辉
宋波涛
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Guizhou Institute Of Biotechnology Guizhou Key Laboratory Of Biotechnology Guizhou Potato Research Institute Guizhou Food Processing Research Institute
Huazhong Agricultural University
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Guizhou Institute Of Biotechnology Guizhou Key Laboratory Of Biotechnology Guizhou Potato Research Institute Guizhou Food Processing Research Institute
Huazhong Agricultural University
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Abstract

本发明公开了马铃薯StGAPC基因在调控马铃薯淀粉含量上的用途，所述马铃薯StGAPC基因的CDS如SEQ ID No.1所示。通过抑制马铃薯StGAPC基因表达提高马铃薯块茎淀粉含量。本发明通过抑制StGAPC基因表达可以提高马铃薯块茎淀粉含量，为提高马铃薯的淀粉含量提供了一个新的方法。

Description

马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途
技术领域
本发明属于基因领域，具体涉及一种马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途。
背景技术
淀粉作为高等植物中碳水化合物主要贮藏形式，同时也是粮食作物产品的最主要成分。影响马铃薯块茎淀粉含量的因素包括光合作用、蔗糖运输、块茎中淀粉的积累、植株本身对淀粉及其代谢物的消耗，调控上述代谢途径的基因均可能对淀粉含量产生影响。马铃薯光合作用固定的碳源和能量通过蔗糖运输蛋白基因(SUT1)进入块茎参与淀粉的合成。淀粉的合成和降解主要涉及腺苷二磷酸葡萄糖(ADPG)焦磷酸化酶(淀粉合成的起始酶，催化葡萄糖-1-p形成ADPG)、淀粉合成酶(将ADPG的葡聚糖基转移到α-1,4-葡萄糖的非还原性末端)、淀粉分支酶(在直链淀粉上形成α-1,6分支点)、淀粉去分支酶(与分支酶的作用相反)、淀粉磷酸化酶和淀粉水解酶(将淀粉降解为葡萄糖单体)。马铃薯细胞质甘油醛-3-磷酸脱氢酶(StGAPCs)是糖酵解反应中的关键酶，在糖酵解过程中参与第一个ATP的形成，催化3-磷酸甘油醛转变为1,3-二磷酸甘油醛，同时产生NADPH，从而使植物体维持较高的能荷水平而正常生长。该酶是一种多功能蛋白，除作为关键酶参与糖酵解等能量代谢外，该酶还参与基因表达的转录后调控、核tRNA运输、DNA复制与修复、组蛋白表达调控、端粒结构调节、细胞凋亡调控等功能，在植物抗旱、抗盐、耐高温等过程中发挥功能。
发明内容
本发明所要解决的技术问题为：如何提供一种与马铃薯淀粉含量相关的基因及调控方法。
本发明的技术方案为：马铃薯StGAPC基因在调控马铃薯淀粉含量上的用途，所述马铃薯StGAPC基因的CDS如SEQ ID No.1所示。
进一步地，通过抑制马铃薯StGAPC基因表达提高块茎淀粉含量。
本发明还公开了一种马铃薯StGAPC基因沉默载体，含有SEQ ID No.2所示的核苷酸序列。
上述所述的沉默载体在提高马铃薯块茎淀粉含量上的应用。
一种马铃薯StGAPC基因沉默载体的构建方法，包括以下步骤：
(1)以含有SEQ ID No.1所示基因的质粒为模板，以SEQ ID No.3和SEQ ID No.4所示核苷酸序列为引物进行PCR扩增；
(2)以步骤(1)扩增的产物为模板，以SEQ ID No.5和SEQ ID No.6所示的核苷酸序列为引物进行PCR扩增；
(3)将步骤(2)的扩增产物与pHellsgate 8载体进行重组，得到含有SEQ ID No.2所示核苷酸片段的马铃薯StGAPC基因沉默载体。
在本发明中，通过农杆菌介导的遗传转化获得抑制表达StGAPC的马铃薯材料，并对抑制表达StGAPC的马铃薯株系块茎淀粉含量鉴定，结果表明阳性植株块茎淀粉含量均显著提高。
与现有技术相比，本发明具有以下有益效果：
本发明通过抑制StGAPC基因表达可以提高马铃薯块茎淀粉含量，为提高马铃薯的淀粉含量提供了一个新的方法。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为从商业渠道购买得到的。
实施例1通过农杆菌介导的遗传转化获得抑制表达StGAPC的马铃薯材料
①制备含StGAPC片段的转化载体，并转化农杆菌
根据StGAPC基因cDNA序列(SEQ ID No.1)选择RNAi干涉片段(SEQ ID No.2)，以含有StGAPC片段的质粒为模板，引物RNAi-GAPC1F：AAAAAGCAGGCTTGTGGGTGTCAACGAGAATGAATAC(SEQ ID No.3)与RNAi-GAPC1R：AGAAAGCTGGGTTCTTACAGTAAGGTCGACAACAGAA(SEQ IDNo.4)，PCR扩增(95℃5min，94℃30s，60℃30s，72℃30s，30个循环，72℃5min)，凝胶电泳回收目的片段(373bp)。以回收的片段为模板，接头引物attB1：GGGGACAAGTTTGTACAAAAAAGCAGGCT(SEQ ID No.5)与attB2：GGGGACCACTTTGTACAAGAAAGCTGGGT(SEQ ID No.6)，PCR扩增(95℃5min，94℃30s，60℃30s，72℃30s，30个循环，72℃5min)获得含有接头序列的片段，凝胶电泳回收目的片段(407bp)。配置TOPO克隆反应体系(PCR产物4μL，盐溶液1μL，pHellsgate 8载体1μL)，轻轻混匀并室温孵育5分钟后置冰上。将DH5α感受态细胞置于冰上解冻，将反应产物加入到100μl感受态细胞中，轻轻混匀，冰上静置30min。42℃水浴热激45s后，立即置于冰上冷却2min。加入900μlLB液体培养基(不添加抗生素)，37℃摇菌1h(转速200rpm)。5,000rpm离心5min，弃上清，用剩余培养基将菌体重悬，用无菌涂布棒在含有25mg/ml壮观霉素的LB固体培养基平板上，37℃培养箱中倒置培养过夜。用灭菌牙签挑取单菌落，在新的含有25mg/ml壮观霉素的LB固体培养基平板上，37℃培养箱中倒置培养过夜。以菌落为模板，引物RNAi-GAPC1F(SEQ IDNo.3)与RNAi-GAPC1R(SEQ ID No.4)，PCR扩增(95℃5min，94℃30s，60℃30s，72℃30s，30个循环，72℃5min)检测。分别挑取3个阳性克隆菌斑，接入30ml含有25mg/ml壮观霉素的LB液体培养基中，37℃200rpm培养过夜，经测序鉴定后获得含StGAPC片段的转化载体。
取-80℃保存的农杆菌感受态插入冰中，至处于冰水混合状态。每100μ1感受态加入1μg质粒DNA，轻轻混匀，依次于冰上静置5分钟、液氮5分钟、37℃水浴5分钟、冰浴5分钟。加入700μL无抗生素的LB液体培养基，于28℃振荡培养2～3小时。6000rpm离心1min收菌，留取100μl左右上清轻轻吹打重悬菌块涂布于含25mg/ml壮观霉素的YEB平板上，倒置放于28℃培养箱培养2-3天。以菌落为模板，引物RNAi-GAPC1F(SEQ ID No.3)与RNAi-GAPC1R(SEQID No.3)，PCR扩增(95℃5min，94℃30s，60℃30s，72℃30s，30个循环，72℃5min)检测。挑取1个阳性克隆菌斑，接入30ml含有25mg/ml壮观霉素的YEB液体培养基中，28℃200rpm培养48h，取菌液加入25％灭菌甘油，于-80℃保存备用。
②获得马铃薯试管薯
将试管苗AC142(马铃薯野生二倍体材料AC142，该材料是在现有技术中广泛使用的一个马铃薯野生种，如在文献“马铃薯高效染色体加倍方法建立与抗寒资源创制，作物学报，2020，46(11)：1659-1666”中公开使用，申请人保证从申请日起20年内向公众发放。)接入蔗糖8％和含活性炭的MS培养基中，培养至试管薯直径约0.5cm。培养条件为温度18～20℃，光强2000lx，光照8h+黑暗16h。
③获得稳定遗传转化的材料
将含有遗传转化载体的农杆菌接种于20-25mL YEB培养基(含转化载体对应抗生素和利福平)中，于28℃、240r/min摇床上培养20-24h，取2mL培养物至50mL YEB培养基中(含转化载体对应抗生素)于28℃、240r/min摇床上培养OD600约0.5。5000g离心6min，其沉淀用20mL MS液体培养基(蔗糖浓度3％)重新悬浮。
将试管薯切成厚度1-2mm的薄片，在前述农杆菌菌液中浸泡约5-10min。将薯片转移至无菌滤纸上，吸干表面菌液，转入共培养培养基(MS基本培养基+3％蔗糖(w/v)+0.2mg/LIAA+0.2mg/L GA3+0.5mg/L 6-BA+2mg/L ZT)中，置于24-26℃暗培箱中暗培养养2d。
转移含转化载体对应抗生素和400mg/L羧苄青霉素的芽分化培养基上，置于光照强度2000lx、光周期16h光照/8h黑暗、温度23±1℃的条件下培养。一周后将其侧芽剪掉以避免营养消耗而不利于抗性芽分化。21-28d后，从试管薯薄片中央长出抗性芽，当抗性芽长达1cm-2cm时，将其切下转入含转化载体对应抗生素和200mg/L羧苄青霉素的MS培养基上生根筛选培养，进而获得完整植株，每个抗性芽作为一个株系单独编号。
④抑制表达StGAPC的马铃薯株系的鉴定
将获得的株系分别扩繁，抽提基因组DNA作为模板，通过PCR扩增及测序，确定转基因株系。获得3个转基因株系，分别记为：Ri-4、Ri-6和Ri-8。
实施例2抑制表达StGAPC的马铃薯株系块茎淀粉含量鉴定
将鉴定为阳性的转基因株系扩繁，待试管苗生长到4-5叶后移栽到隔离大棚。待马铃薯成熟后，收获块茎黑暗储藏。待块茎休眠期结束后，选择薯重10-20g，芽长在1cm左右的块茎大棚盆栽。栽培基质为每5kg椰砖加水泡发后，与10kg商品有机肥、0.5kg复合肥(N:P:K＝15:15:15)混匀，分装到直径30cm的塑料花盆中。每盆种植1株，每个材料18株。待马铃薯成熟后，收获块茎，选择无病虫害与机械损伤的块茎，测定其淀粉含量变化，结果如表1所示。
表1马铃薯株系淀粉含量
Figure BDA0003482409010000041
从表1看出，抑制StGAPC基因表达后，3个转基因株系块茎淀粉含量提高了17.6％、26.1％与34.52。
序列表
<110> 贵州省生物技术研究所（贵州省生物技术重点实验室、贵州省马铃薯研究所、贵州省食品加工研究所） 华中农业大学
<120> 马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1393
<212> DNA
<213> Solanum tuberosum
<400> 1
cccctcactg tcacgtgact ccccctcccc cctaacaccc ctttatattc cgcactacag 60
ccatacattt cttgctacat tttgactgtt gtttacacta taccctaatt tctctcttta 120
catattttct tagctttcac cctttttttc ttagcaatgg ccaatggcaa gatcaaaatc 180
ggaatcaacg gattcggtag aattggtcgt ttggttgcta gagttgctct gcagagggat 240
gatgttgaac tagttgcagt gaatgatcca tttatttcca ctgattacat gacatatatg 300
tttaaatatg attcagtaca tggacaatgg aagcaccatg agcttaaggt taaggatgag 360
aagacccttc tcttcggtga gaaggctgtt acagtttttg gaatcaggaa ccctgaagat 420
atcccatggg gggaagctgg tgctgacttc gtcgttgaat caaccggtgt cttcactgac 480
aaggacaagg ctgctgctca cttgaagggt ggtgccaaga aggttgtgat ctccgctcct 540
agcaaagatg cccccatgtt tgttgtgggt gtcaacgaga atgaatacaa gccagagttg 600
gacattgtct ccaacgctag ttgcactact aactgccttg cacctttggc taaggtcatc 660
aatgacaggt ttggcattgt ggagggtctc atgaccactg tccacgccat gactgccacc 720
cagaaaactg ttgatggtcc atccatgaag gactggagag gtggaagagc tgcttcattc 780
aacatcatcc ctagcagcac tggagcagcc aaggctgttg gaaaagtgct cccatcactt 840
aacggcaaat tgactggaat ggccttcaga gtaccaactg ctgatgtttc tgttgtcgac 900
cttactgtaa gacttgagaa agaagcctca tatgaagaca tcaaggctgc aatcaaggag 960
gaatcagagg gtaaattgaa gggtatcttg ggatacactg aagatgatgt ggtttccaca 1020
gactttgttg gtgacagcag gtcaagcatt ttcgatgcca aggctggaat tgctttgagc 1080
aagaactttg tgaaacttgt gtcatggtat gacaacgaat ggggttacag ttcccgtgtg 1140
attgatttga tctgccatat ggccaaggct tgattgatgc tgctggggag cagaagacaa 1200
tctgttttag tttttgcttg aagtattagt tttctgggcc gggagtggtc tttcttgttt 1260
atgtgtaatg gaataaccag agaggaacgg aaccctgttg ttatctttga ggatatcttt 1320
tactgtttga cttgtcatga atgaatcaaa ctttactttt tccagtatca gtcatcagtt 1380
tcttgccttt tgt 1393
<210> 2
<211> 349
<212> DNA
<213> Solanum tuberosum
<400> 2
tgtgggtgtc aacgagaatg aatacaagcc agagttggac attgtctcca acgctagttg 60
cactactaac tgccttgcac ctttggctaa ggtcatcaat gacaggtttg gcattgtgga 120
gggtctcatg accactgtcc acgccatgac tgccacccag aaaactgttg atggtccatc 180
catgaaggac tggagaggtg gaagagctgc ttcattcaac atcatcccta gcagcactgg 240
agcagccaag gctgttggaa aagtgctccc atcacttaac ggcaaattga ctggaatggc 300
cttcagagta ccaactgctg atgtttctgt tgtcgacctt actgtaaga 349
<210> 3
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaaagcagg cttgtgggtg tcaacgagaa tgaatac 37
<210> 4
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
agaaagctgg gttcttacag taaggtcgac aacagaa 37
<210> 5
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggggacaagt ttgtacaaaa aagcaggct 29
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggggaccact ttgtacaaga aagctgggt 29

Claims (5)

1.马铃薯StGAPC基因在调控马铃薯淀粉含量上的用途，所述马铃薯StGAPC基因的CDS如SEQ ID No.1所示。
2.根据权利要求1所述的用途，其特征在于，通过抑制马铃薯StGAPC基因表达来提高马铃薯块茎淀粉含量。
3.一种马铃薯StGAPC基因沉默载体，含有SEQ ID No.2所示的核苷酸序列。
4.权利要求3所述的一种马铃薯StGAPC基因沉默载体的构建方法，包括以下步骤：
(1)以含有SEQ ID No.1所示基因的质粒为模板，以SEQ ID No.3和SEQ ID No.4所示核苷酸序列为引物进行PCR扩增；
(2)以步骤(1)扩增的产物为模板，以SEQ ID No.5和SEQ ID No.6所示的核苷酸序列为引物进行PCR扩增；
(3)将步骤(2)的扩增产物与pHellsgate 8载体进行重组，得到含有SEQ ID No.2所示核苷酸片段的马铃薯StGAPC基因沉默载体。
5.权利要求3所述的沉默载体在提高马铃薯块茎淀粉含量上的应用。
CN202210071575.7A 2022-01-21 2022-01-21 马铃薯StGAPC基因在提高马铃薯淀粉含量上的用途 Pending CN114703208A (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011375A1 (en) * 1990-12-21 1992-07-09 Amylogene Hb Genetically engineered modification of potato to form amylose-type starch

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011375A1 (en) * 1990-12-21 1992-07-09 Amylogene Hb Genetically engineered modification of potato to form amylose-type starch
AU9109791A (en) * 1990-12-21 1992-07-22 Amylogene Hb Genetically engineered modification of potato to form amylose-type starch
EP0563201A1 (en) * 1990-12-21 1993-10-06 Amylogene Hb Genetically engineered modification of potato to form amylose-type starch
PL169859B1 (pl) * 1990-12-21 1996-09-30 Amylogene Hb Sposób hamowania tworzenia sie w ziemniakach skrobi typu amylopektyny PL

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GENBANK: AF527779.1: "Solanum tuberosum glyceraldehyde 3-phosphate dehydrogenase mRNA, complete cds" *
MOHAMMAD-REZA HAJIREZAEI 等: "The influence of cytosolic phosphorylating glyceraldehyde3-phosphate dehydrogenase (GAPC) on potato tuber metabolism" *

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