CN114891804B - Tst1基因在增强马铃薯低温糖化抗性中的应用 - Google Patents
Tst1基因在增强马铃薯低温糖化抗性中的应用 Download PDFInfo
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Abstract
本发明提供了TST1基因在增强马铃薯低温糖化抗性中的用途。马铃薯TST1基因CDS序列包含2214bp核苷酸。通过沉默TST1能显著降低马铃薯块茎低温贮藏后的还原糖的累积，提高块茎炸片品质，并且有效降低有害物质丙烯酰胺的含量，说明抑制TST1的马铃薯块茎在抗低温糖化方面效果极其显著，是一个在马铃薯抗低温糖化应用上的功能基因。
Description
技术领域
本发明属于马铃薯分子育种领域，具体涉及TST1基因在增强马铃薯低温糖化抗性中的应用。
背景技术
马铃薯是世界继水稻，小麦，玉米后的第四大粮食作物。是我国极为重要的粮菜兼用作物，我国马铃薯种植面积和产量均居世界第一，约占四分之一。马铃薯在保障世界和我国粮食安全中具有十分重要的作用。
马铃薯收获后的块茎为了延长货架期，减少贮藏过程中的发芽，病害和水分流失导致的品质下降。通常将马铃薯块茎在低温下贮藏，但是低温会刺激块茎淀粉分解，导致还原糖在块茎中积累，称为低温糖化。低温糖化是马铃薯加工业中长期存在的关键问题。低温糖化后的马铃薯块茎在高温油炸加工过程中下，还原糖会与游离氨基酸(主要是天冬酰胺)发生非酶促美拉德反应，使得油炸加工品如炸薯条和薯片变黑变苦，并伴随从而产生具有潜在致癌性的丙烯酰胺，严重限制马铃薯的加工品质。然而，目前有关增强马铃薯低温糖化抗性方法的研究仍有待加强。
在植物中，液泡是可溶性糖累积的主要亚细胞器。而可溶性糖运载至液泡需要一类糖转运蛋白的介导。目前植物中已经鉴定出多类糖转运蛋白，如液泡膜糖转运蛋白(TSTs)，液泡葡萄糖转运蛋白(VGTs)，SUC4型蔗糖转运蛋白和三个SWEET蛋白家族成员AtSWEET2、AtSWEET16和AtWEET17。在这些糖转运蛋白中，据报道只有TST类的糖转运蛋白在库器官中起作用。马铃薯低温糖化过程中，淀粉的降解产物在细胞质中合成蔗糖，蔗糖进一步由糖转运蛋白转运至液泡中，并且在液泡酸性转化酶的作用下分解为还原性的葡萄糖和果糖，从而导致低温糖化的发生。但是目前在马铃薯中关于通过抑制蔗糖由细胞质向液泡中的转运而抑制低温糖化还未见报道，因此鉴定低温糖化过程中参与蔗糖转运调控的液泡膜糖转运蛋白，并明确其对低温糖化的影响兼具很强的生物学和经济意义。
基于此，发明人采用序列比对结合荧光定量分析筛选到马铃薯块茎低温贮藏过程中高表达的液泡膜糖转运蛋白TST1。
RNA干扰(RNAi)指小的双链RNA通过刺激互补靶mRNA的特异性降解来抑制靶基因的表达，引起表型的变化，进而根据表型变异研究目标基因的功能。与基因敲除的方法相比，RNAi能抑制当代植物的基因表达，从而实现对目标基因进行沉默和功能分析，已广泛应用于马铃薯、烟草和番茄等的研究。
因而，发明人在马铃薯中利用RNAi技术沉默TST1,以研究TST1基因沉默对马铃薯生长发育有无影响以及经过沉默后马铃薯块茎的低温糖化抗性。结果，发明人惊奇地发现，沉默植株没有发育表型，表明TST1基因沉默后对植物生长发育等基本过程没有太大影响，适合育种。进一步，发明人通过一系列方法研究发现，TST1沉默的块茎低温贮藏后，累积的还原性的含量极显著降低，油炸加工品质明显改善，并且丙烯酰胺的含量显著降低，证明沉默TST1能增强马铃薯块茎低温糖化抗性。
发明内容
本发明要解决的关键技术问题在于TST1基因在增强马铃薯低温糖化抗性中的应用。为解决上述技术问题，本发明采用如下技术方案：
1.马铃薯TST1基因，CDS序列如序列表SEQ ID No.1所示，包含2214bp核苷酸。干涉TST1所用序列如序列表SEQ ID No.2所示。
2.马铃薯TST1基因植物干涉载体构建方法，包括：马铃薯栽培种E3的cDNA中PCR得到干涉序列，其中，所用PCR引物为正向引物：5'—AAAAAGCAGGCTGCAGGGTTGGGATAATGCT—3'；反向引物：5'—AGAAAGCTGGGTTAGGGGGACCAGAGTAACCG—3'；干涉TST1所用序列为序列表SEQID No.2所示。将上述PCR产物进行回收。以此为模板，利用通用引物：attBl、attB2，进行第二轮PCR扩增，胶回收产物通过BP反应重组到pDNOR221载体，阳性克隆pDNOR221-TST1通过LR反应重组到pHellsgate8载体，热击转化大肠杆菌DH5a，并测序验证。测序正确阳性克隆pHellsgate8-TST1电击转化农杆菌GV3101，PCR检测为阳性的克隆进行保存，用于进一步的遗传转化。
3.马铃薯TST1基因在增强马铃薯低温糖化抗性中的应用验证方法，包括：(1)马铃薯遗传转化，(2)转基因阳性株系检测，(3)转基因马铃薯块茎低温糖化抗性鉴定。
4.马铃薯TST1基因在增强马铃薯低温糖化抗性中的应用，所述应用通过干扰、下调TST1基因表达，或通过编辑TST1基因沉默突变予以实现。
5.马铃薯TST1基因在降低马铃薯块茎还原糖含量中的应用，所述应用通过干扰、下调TST1基因表达，或通过编辑TST1基因沉默突变予以实现。
6.马铃薯TST1基因在降低马铃薯块茎炸片色泽中的应用，所述应用通过干扰、下调TST1基因表达，或通过编辑TST1基因沉默突变予以实现。
有益效果：沉默TST1能显著降低马铃薯块茎低温贮藏后的还原糖的累积，提高块茎炸片品质，并且有效降低有害物质丙烯酰胺的含量，说明抑制TST1的马铃薯块茎在抗低温糖化方面效果极其显著，是一个在马铃薯抗低温糖化应用上的功能基因。
附图说明
图1：干涉载体pHellsgate8-TST1构建过程示意。
图2：干涉转基因株系TST1的表达量检测。
图3：TST1沉默的马铃薯块茎的抗低温糖化研究结果，其中，3A：转基因株系块茎低温贮藏4℃30d的炸片图片；3B：转基因株系块茎低温贮藏4℃30d的还原糖含量；图3C：转基因株系块茎低温贮藏4℃30d的油炸薯片丙烯酰胺含量。
具体实施方法
本发明专利下述实施例中使用方法和装置，如无特殊说明，均为常规方法和装置；所用器材、试剂均为试剂公司购买的常规器材和试剂。为使本发明专利的目的、技术方案和优点更加清楚，下面结合具体实施例对本发明专利的具体实施方式进行详细说明。这些优选实施方式的示例在具体实施例中进行了例示。在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明专利的技术方案，在实施例中仅仅示出了与根据本发明专利的方案密切相关的技术方案和/或处理步骤，而省略了关系不大的其他细节。
实施例1
本实施例提供马铃薯TST1基因，包括：
马铃薯TST1基因，CDS序列如序列表SEQ ID No.1所示，包含2214bp核苷酸。干涉TST1所用序列如序列表SEQ ID No.2所示。
实施例2
本实施例提供马铃薯TST1基因植物干涉载体构建方法，包括：
发明人自马铃薯栽培种(Solanum tuberosum,简称St)E3的cDNA中PCR得到干涉序列，其中，所用PCR引物为：正向引物：5'—AAAAAGCAGGCTGCAGGGTTGGGATAATGCT—3'；反向引物：5'—AGAAAGCTGGGTTAGGGGGACCAGAGTAACCG—3'
干涉TST1所用序列为：GCAGGGTTGGGATAATGCTACTATTGCTGGAGCTGTTGTTTACATAAAGAAGGAGCTTGCTTTGGATGCTTCAGTGGAAGGACTTGTTGTTGCGATGTCACTCATTGGAGCCACACTTGTCACAACTTGTTCTGGATCCATAGCTGACAGTATTGGTCGTCGCCCTATGCTTATTATGTCATCCATGCTTTATTTCCTTAGTGGCTTAATAATGTTGTGGTCCCCTAATGTCTATGTCCTGCTTATAGCTAGACTATTAGATGGATTTGGAATCGGCTTAGCGGTTACTCTGGTCCCCCTA(SEQ ID NO:2)。
将上述PCR产物进行回收。以此为模板，利用通用引物：attBl(5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3')、attB2(5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3')，进行第二轮PCR扩增，胶回收产物通过BP反应(目的片段1.4μl，pDNOR221质粒lμl，BP酶0.6μl，25℃重组16h，加0.3μl蛋白酶K，37℃反应10min)重组到pDNOR221载体，阳性克隆pDNOR221-TST1)通过LR反应(入门载体1.2μL，phellsgate8 1.2μL，LR酶0.6μL，25℃重组16h，加0.3μL蛋白酶K，37℃反应10min。)重组到pHellsgate8载体，热击转化大肠杆菌DH5α，并测序验证。测序正确阳性克隆pHellsgate8-TST1电击转化农杆菌GV3101，PCR检测为阳性的克隆进行保存，用于进一步的遗传转化。
实施例3
本实施例提供干涉马铃薯TST1基因在增强马铃薯低温糖化抗性中的应用，包括：
1.马铃薯遗传转化
遗传转化将pHellsgate8-TST1的农杆菌GV3101接种于加50mg/L Spe和50mg/LRif的YEB培养基中，于28℃、200r/min摇床上培养至0D600为0.6左右。4000r/min离心6min，其沉淀用3％MS液体培养基重新悬浮。将生长12-16周、直径0.5cm左右的E3试管薯切成1-2mm厚的薄片，在上述农杆菌菌液中浸泡10min，每隔5min摇一次。浸染结束后取出薯片，用无菌滤纸吸干表面菌液。转入共培养培养基S1:3％MS+1(mg/L)IAA+0.2(mg/L)GA3+0.5(mg/L)6-BA+2(mg/L)ZT培养皿中，于23℃暗培养2d后，转到S2培养基：S1+75mg/LKam+400mg/LCef+200mg/L Tim中，置于光照强度2000lux，光周期16h/d，温度23℃的条件下培养直到抗性芽再生。当抗性芽长达0.5-1cm时，将其切下转入生根培养基S3:3％MS+100mg/L Kam+400mg/L Cef+200mg/L Tim筛选。对生根的芽进行DNA提取，35S通用引物PCR检测转基因阳性植株。转基因培养基见表1。
表1马铃薯转基因培养基
2.转基因阳性株系检测
在超净工作台中剪取待测苗1-2个小叶片于2ml离心管中。用CTAB法提取DNA。DNA提取完成后，以其作为模板进行PCR转基因株系检测，使用载体引物35s：5'-GACGCACAATCCCACTATCC-3’，反向引物为基因引物：StTST1-R:5'-TAGGGGGACCAGAGTAACCG-3’，用PCR进行扩增，电泳检测获得3个转基因植株。利用植物RNA快速提取试剂盒(庄盟)进行RNA的提取转基因植株和对照植株总RNA，然后，利用RT MasterMix with AccuRT(ABM)试剂盒反转录产生cDNA。以cDNA作为模板，利用定量引物对目标基因TST1进行表达量检测，结果显示3个阳性转基因株系中TST1表达量均被大幅抑制(图2)。
3.转基因马铃薯块茎低温糖化抗性鉴定
TST1转基因功能鉴定发明人将3个TST1干涉转基因株系和对照(转基因所用的受体材料E3)同时种植在同一个温室大棚中，相同的管理条件，每个株系种植18盆，植株长势良好，薯块成熟度高，转基因株系在田间农艺性状上与对照无明显差异。收获转基因株系块茎常温20℃避光保存7-10天后，用来炸片和还原性糖测定。块茎低温4℃避光处理30d后选取3个大小均匀(大于50g)的薯进行取样。测糖的每个薯纵切一分为二，薯的一半用来炸片，炸片所用的工艺流程是：原料马铃薯--清洗--去皮--切片--漂洗一一脱水--油炸(普通调和油，恒温170℃，3min)。薯的另一半用来测定还原糖，每个时间点取3个生物学重复。
首先对TST1转基因株系块茎4℃贮藏30d进行了油炸加工品质色泽指数，直观的结果显示转基因株系的炸片色泽明显的浅于对照(图3A)。此结果表明干涉TST1对马铃薯块茎低温糖化起到了非常明显的抑制作用，使得原本不适用于低温贮藏后炸片加工的栽培种E3获得了低温贮藏l个月后仍能达到炸片色泽较浅的能力。
还原糖的含量测定结果显示转基因株系与对照的差异更为显著，与对照相比，低温贮藏下TST1干涉株系的各株系大多比对照明显低，3个转基因株系还原糖含量下降程度均超过80％以上(图3B)，即低温下转基因株系中还原糖的积累速率非常慢，这也是炸片色泽浅的主要直接原因。同样对油炸薯片丙烯酰胺含量的测定也显示3个转基因的油炸薯片丙烯酰胺含量极显著低于对照(图3C)。试验检测结果证明，沉默TST1的马铃薯块茎在抗低温糖化方面效果极其显著，是一个在马铃薯抗低温糖化应用上的功能基因。
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。
<110> 华中农业大学
<120> TST1基因在增强马铃薯低温糖化抗性中的应用
<160> 2
<210> 1
<211> 2214bp
<212> DNA
<213> 马铃薯（Solanum tuberosum）
<400> 1
1 atgaatggtg ctgtgttagt tgcgcttgcc gcgacaattg gaaacttttt gcagggttgg
61 gataatgcta ctattgctgg agctgttgtt tacataaaga aggagcttgc tttggatgct
121 tcagtggaag gacttgttgt tgcgatgtca ctcattggag ccacacttgt cacaacttgt
181 tctggatcca tagctgacag tattggtcgt cgccctatgc ttattatgtc atccatgctt
241 tatttcctta gtggcttaat aatgttgtgg tcccctaatg tctatgtcct gcttatagct
301 agactattag atggatttgg aatcggctta gcggttactc tggtccccct atatatatct
361 gagactgctc catcagaaat acgagggtca ctgaatactc ttccacaatt cactggttct
421 ggtggaatgt tcttggccta ctgcatgatt ttcggaatgt ccttaatgac agcgccgagt
481 tggcgattga tgttgggtgt tctttcaatt ccttctctta tctatttcgt attagttgta
541 ctttatttgc ctgagtctcc tcgatggcta gtgagtaaag gaagaatggt cgaggcaaaa
601 caagttttgc agaaattgcg tggcatagag gatgtttcag gggagatggc attgcttgtt
661 gagggtttgg cagttggcat tgaaccatca atagaagagt atatcattgg tccagctaat
721 gcgcttactg aggatcagga cctggctact gacaaagatc atatcaagtt gtacggccca
781 gaggaaggcc tctcgtgggt ggccaagcca gttactggac agagtagtct agctcttgtg
841 tccaggcaag ggagcatggt gcagcagagt gtgcctctta tggatcctct tgtgactcta
901 tttggtagtg tccatgagaa tctccctgat acaggaagta tgagaagcat gctattcccc
961 aatttcggaa gcatgatcag caccatggat cctcatgtca aagatgatca ctgggatgag
1021 gagagtctgc agagagaagg tgatgattat ccttcggatg gcggtgcaga ttctgatgac
1081 aatctacaaa gtccattgat atcacgtcaa acaaccgctg tggaaaccgt agtccctcat
1141 ccccatggca gcactctgag cgtgaggcgg catagcagcc ttatgcaagg caatgctgga
1201 gagggtgtag gcagcatggg cattggtggt ggttggcagt tggcatggaa atggtctgaa
1261 agggaaggtg aagatggaac taaagaagga ggcttcaaaa ggatatattt gcatcaggag
1321 gcaggccctg gctctcgacg tgggtctctc gtttcagtcc ctggtggtga tattcctgaa
1381 gatggtgaat tcatacaagc tgcggctttg gtaagtcagc ctgcacttta ctcaaaggaa
1441 cttatggatc agcatcctgt gggaccagcg atggtccatc catctgaaac tgcttcaaaa
1501 ggtccgagtt gggctgctct tcttgaacct ggagtcaagc gagcgctcat tgttggaatt
1561 ggaattcaaa tattgcaaca gttttccggt ataaatggag tcatgtacta cactcctcaa
1621 atccttgagc aggcaggtgt aggagttctt ctttctaact ttggcatcgc atcagactca
1681 gcatccttcc tcatcagtgc gttaacaaac ttcctgatgc tcccttctgt agctattgca
1741 atgcgattca tggatgtggc tggcagaagg tcgctgctgc tgtacactat tcctgttctc
1801 atactatcac tcatctgtct tgtcattggt aacactatca acctcgggag tgtggctcat
1861 gcagtcgttt ctactatttg cgtgattctc tacttttgct tctttgtaac gggctatgga
1921 ccaatcccaa atattctctg ctcagaaatt ttcccaacaa gggttcgtgg tttgtgcatt
1981 gccatctgtg ctctcgtctt ctggatatgt gatgtcattg tgacttacac actgcctgtg
2041 atgctcaact cgattggctt atctggagtt tttggtattt atgccattgt atgtgtcatt
2101 tcttggattt tcgtcttctt gagggttccc gaaaccaaag gcatgccctt agaagtcatt
2161 acagagttct ttgctgttgg tgcaagacaa gctgctatcg cgaagcatga gtag
<210> 2
<211> 301
<212> DNA
<213> 马铃薯（Solanum tuberosum）
<400> 2
1 gcagggttgg gataatgcta ctattgctgg agctgttgtt tacataaaga aggagcttgc
61 tttggatgct tcagtggaag gacttgttgt tgcgatgtca ctcattggag ccacacttgt
121 cacaacttgt tctggatcca tagctgacag tattggtcgt cgccctatgc ttattatgtc
181 atccatgctt tatttcctta gtggcttaat aatgttgtgg tcccctaatg tctatgtcct
241 gcttatagct agactattag atggatttgg aatcggctta gcggttactc tggtccccct
301 a
Claims (3)
1.TST1基因在增强马铃薯低温糖化抗性中的应用，其特征在于所述TST1基因CDS序列如序列表SEQ ID No.1所示；所述应用是指干扰TST1基因表达实现增强马铃薯低温糖化抗性，干扰表达所用片段如序列表SEQ ID No.2所示。
2.马铃薯TST1基因在降低马铃薯块茎还原糖含量中的应用，其特征在于所述TST1基因CDS序列如序列表SEQ ID No.1所示；所述应用是指干扰TST1基因表达实现降低马铃薯块茎还原糖含量，干扰表达所用片段如序列表SEQ ID No.2所示。
3.马铃薯TST1基因在降低马铃薯块茎炸片色泽中的应用，其特征在于所述TST1基因CDS序列如序列表SEQ ID No.1所示；所述应用是指干扰TST1基因表达实现降低马铃薯块茎炸片色泽，干扰表达所用片段如序列表SEQ ID No.2所示。
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