CN112760304A - 基于基因编辑技术的gbssi突变型蛋白及其在植物育种中的应用 - Google Patents
基于基因编辑技术的gbssi突变型蛋白及其在植物育种中的应用 Download PDFInfo
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- CN112760304A CN112760304A CN202110239257.2A CN202110239257A CN112760304A CN 112760304 A CN112760304 A CN 112760304A CN 202110239257 A CN202110239257 A CN 202110239257A CN 112760304 A CN112760304 A CN 112760304A
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Abstract
本发明公开了水稻直链淀粉合成酶GBSSI突变型蛋白、其编码基因及在育种中的应用，所述GBSSI突变型蛋白的氨基酸序列存在以下突变：其对应的水稻GBSSI氨基酸序列的第178位氨基酸发生突变，本发明还公开了利用基因编辑创制直链淀粉含量5％左右水稻的育种方法。本发明利用CRISPR/Cas9基因编辑技术，对Waxy基因进行定点突变，通过后代筛选，在T1代可以获得剔除Cas9元件的新材料，而新材料的基本农艺性状与野生型相比无明显改变。相对于传统的化学诱变育种和杂交育种等手段，基因编辑定向改良分子育种技术具有高效、精准等优点，极大提高育种效率、加快育种进程。
Description
技术领域
本发明涉及水稻遗传育种、糯性品质改良邻域，具体地，本发明涉及基于基因编辑技术的GBSSI突变型蛋白及其在植物育种中的应用。
背景技术
水稻是重要的粮食作物，随着人们生活水平的提高，对优质水稻品种的需求进一步增大。水稻直链淀粉含量影响水稻糯性，进而影响稻米的品种和口感。高直链淀粉稻米的糊化温度较高，胶稠度降低，煮成米饭的黏性、光泽度及柔软性都比较差；低直链淀粉含量的稻米，煮成饭后米粒晶莹、口感软糯，从而备受人们青睐。因而，通过降低水稻中的直链淀粉含量从而来改善稻米品质一直以来是育种家的目标。
水稻中直链淀粉主要由水稻蜡质基因Waxy(Wx)编码的颗粒结合淀粉合成酶(GBSSI)催化合成。研究表明Waxy是控制稻米直链淀粉含量的主效基因，目前大部分基于水稻品质的分子遗传育种都是围绕该基因进行的。在栽培稻中存在三个主要的Wx等位基因，Wxa、Wxb和wx。wx存在于糯稻品种中，是Wx基因的功能缺失形式，wx稻米中没有直链淀粉或者直链淀粉含量非常低(小于2％)。Wxa等位基因存在于大多数籼稻品种中，而大多数粳稻品种含有Wxb等位基因。这两个等位基因之间的主要差异是G/T多态性，该多态性产生的差异剪接会影响Wx mRNA稳定性，导致Wxa等位基因产生的mRNA和蛋白质水平比Wxb高10倍，从而使籼稻产生更多的直链淀粉(一般大于25％，有的在30％以上)，而粳稻中直链淀粉含量一般为15-18％。除了这三个等位基因，近年来通过对一些自然存在的一些直链淀粉含量变化的品种的研究，也发现了其它一些复等位基因，如Wxin、Wxop、Wxmq及Wxmp等。这些等位基因一般在序列上有一个或几个碱基的差异，这些差异会影响Wx基因的表达及GBSS的活性，从而导致直链淀粉含量及稻米品质的不同。
通过传统的育种方法也可以这些优良性状转育到当地品种中，但这种方式耗时、费力、不确定因素较多，很难在短期内对本地品种进行改良。因而需要发展新的技术和方法来加速优良品种的培育。近年来，基因编辑技术，特别是CRISPR/Cas9技术的出现，为快速培育优良新品种提供了可能。本发明通过CRISPR/Cas9技术对水稻Waxy基因进行了人工突变，并获得了直链淀粉含量5％左右的新材料，为水稻新品种改良提供基础材料。
发明内容
本发明的第一个目的是提供一种水稻GBSSI突变型蛋白，所述GBSSI突变型蛋白的氨基酸序列存在以下突变：其对应于水稻GBSSI氨基酸序列的第178位氨基酸发生突变。
本发明首次发现了该178位氨基酸由苏氨酸突变为异亮氨酸并改变稻米直链淀粉含量。本发明的第178位氨基酸的突变还可以包括谷氨酸、甘氨酸、色氨酸、天冬氨酸、色氨酸、丙氨酸、缬氨酸、亮氨酸、脯氨酸、苯丙氨酸、酪氨酸、丝氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、赖氨酸、精氨酸、组氨酸以及终止密码子等19种变异类型。而关于上述的氨基酸的其他变异或提前终止是否影响淀粉合成酶活性、生理功能以及是否改变稻米直链淀粉含量，还有待进一步研究证实。
进一步地，本发明所述的水稻GBSSI突变型蛋白，其特征在于，包括：
(a)其氨基酸序列如SEQ ID NO.2所示；或
(b)在(a)中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具有直链淀粉合成酶活性的由(a)衍生的蛋白质。
本发明第二个目的是提供编码上述突变型蛋白的基因。所述基因可以包括：在严格条件下与编码上述突变型蛋白的核苷酸序列杂交且编码具有直链淀粉合成酶活性的蛋白质的核苷酸序列。
进一步地，所述基因是：
a)其核苷酸序列如SEQ ID NO.1所示；或
b)在严格条件下与a)限定的核酸序列杂交且编码具有直链淀粉合成酶活性蛋白质的核酸序列；
本发明第三个目的是提供一种表达盒、重组载体或细胞，所述表达盒、重组载体或细胞含有编码上述突变型蛋白的基因。除重组载体外，还可以是表达盒，细胞等。
本发明第四个目的是提供上述水稻GBSSI突变型蛋白、基因、表达载体在水稻育种中的应用，特别是水稻糯性品质方面的应用。
本发明第五个目的是提供一种获得低直链淀粉含量(本发明所述低直链淀粉是指直链淀粉含量为5％左右)的水稻的方法，包括如下步骤：
1)使水稻植株包含上述编码GBSSI突变型蛋白的基因；或
2)使水稻植株表达上述水稻GBSSI突变型蛋白。
本发明的最后一个目的是提供一种利用基因编辑技术低直链淀粉含量水稻的育种方法，包括以下步骤：
1)设计Waxy基因定点编辑的靶位点：基因编辑的靶位点核苷酸序列如SEQ IDNO.5所示；
2)构建含有目的片段的CRISPR/Cas9基因编辑载体：
A)靶点接头制备：采用接头引物用双蒸水溶解成母液，母液稀释后90℃30s(second，秒)，移至室温冷却完成退火，即获得靶点接头；
B)将退火获得的靶点片段连接到CRISPR/Cas9表达载体上获得连接产物；
C)将步骤B)的连接产物进行热激法转化大肠杆菌获得重组菌，提取通过验证的含目的条带的菌液的阳性质粒；
3)将阳性质粒转化农杆菌EHA105，获得T0代转基因植株，以引物Waxy TXT-F和Waxy TXT-R对T0代转基因植株进行扩增并测序鉴定获得具备权利要求1或2所述突变型蛋白的植株。
进一步地，所述育种方法还包括将具有突变型蛋白的T0代转基因植株的含有目标等位基因纯合突变的T1代植株的T-DNA载体的剔除，所述T-DNA载体包括筛选标记HPT基因和Cas9元件。
其中，所述T-DNA载体的剔除通过对含有目标等位基因纯合突变的T1代植株的HPT基因和Cas9元件的同时检测，重复多次，筛选得到不携带HPT基因和Cas9元件的T1代单株即为目标植株。
其中，所述HPT基因基因检测方法通过以目标等位基因纯合突变的T1代植株的基因组DNA为模板，以hyg283-F和hyg283-R为引物进行PCR扩增，同时，所述Cas9元件的检测方法通过以目标等位基因纯合突变的T1代植株的基因组DNA为模板，以Cas9 TXT-F和Cas9TXT-F为引物进行PCR扩增，当未同时检测到HPT基因和Cas9元件，表明这成功剔除了T-DNA载体。
有益效果：相对于现有技术，本发明具备以下优点：
1)本发明利用CRISPR/Cas9基因定点编辑技术，对Waxy基因进行编辑，通过后代的筛选，在T1代就可以获得剔除Cas9元件的稳定遗传的材料，而新材料的基本农艺性状无明显改变。相对于化学诱变、杂交转育等育种，基因编辑定向改良分子育种技术具有快速、精准、高效等优点，利用基因功能标记进行基因型选择，将会极大提高育种效率，大大加快育种进程。
2)本发明的基因编辑技术育种，通过对水稻GBSSI蛋白第178位氨基酸的改变，得到的水稻品种的稻米经测定淀粉含量为5％左右。
附图说明
图1显示基因编辑载体Anc689BEmax-WaxysgRNA-Cas9的整个图谱。
图2显示秀水134和基因编辑株系Waxy基因编辑位点测序图。
图3显示秀水134和基因编辑株系GBSSI氨基酸比较图。
图4显示稻米外观示意图。
图5显示秀水134和waxy-m突变体植株的表型观察；其中，图5A为秀水134和waxy-m突变体植株整株表型；图5B为秀水134和waxy-m突变体植株株高统计；图5C为秀水134和waxy-m突变体植株分蘖数统计结果；图5D为秀水134和waxy-m突变体植株穗形；图5E为秀水134和waxy-m突变体植株粒宽；图5F为秀水134和waxy-m突变体植株粒长。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。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材料、试剂等，如无特殊说明，均为市售购买产品。
本发明选取的背景材料为秀水134(是常规水稻品种，存在于本实验室)，该品种是由嘉兴市农业科学研究院、中国科学院遗传与发育生物学研究所、浙江嘉兴农作物高新技术育种中心和余姚市种子管理站所选育的迟熟中粳新品种，全生育期152.2天左右，适宜上海地区种植，具有优良的综合农艺性状，已在生产上大面积推广应用，深受市场欢迎。秀水134株型较紧凑，茎秆粗壮，生育期适中，穗型较大，抗稻瘟病，直链淀粉含量为14％-16％左右，稻米外观透明。本发明通过CRISPR/Cas9基因编辑技术对秀水134的Waxy基因进行定点编辑，获得直链淀粉含量5％左右的突变体，为培育不同水稻糯性品种提供材料。
实施例1：5％左右直链淀粉含量的水稻突变体waxy-m获取过程
1、CRISPR/Cas9修饰靶点的选择
针对Waxy全基因组序列进行扫描，Waxy基因共有13个外显子，根据已有研究得知Waxy单碱基突变位点主要集中在4-6外显子，基于此，我们将突变位点选择限定在第4个外显子上，借助CRISPR-GE(http://skl.scau.edu.cn)以及CRISPR-P(http://crispr.hzau.edu.cn/CRISPR)网站筛选到了第4外显子上的靶向序列：AAGACCGGTGAGAAGATCTA(SEQ ID NO.5)，位于第4外显子中的第10-29位碱基。
2、CRISPR/Cas9载体构建
针对该靶向序列合成以下寡核苷酸：sgRNA-Waxy-F：5’-TGTGTGAGACCGGTGAGAAGATCTA-3’(SEQ ID NO.6)；sgRNA-Waxy-R：5’-AAACTAGATC TTCTCACCGGTCTCA-3’(SEQ ID NO.7)；将合成的引物sgRNA-Waxy-F和sgRNA-Waxy-R用双蒸水溶解，浓度为10μmol母液。将溶解的引物按如下比例混合：8μL双蒸水+1μL sgRNA-F+1μL sgRNA-F。并进行退火反应，获得带接头的双链DNA片段。用BsaI酶切Anc689BEmax-sgRNA-Cas9载体(参见MuguiWang等人,Optimizing base editors for improved efficiency and expanded editingscope in rice,Plant Biotechnology Journal(2019)17:1697-1699)，琼脂糖凝胶电泳并回收酶切的线性载体片段。将酶切回收的载体与退火形成的带粘性末端的双链DNA进行连接反应，连接产物转化大肠杆菌，鉴定阳性克隆并测序验证，获得构建正确的Anc689BEmax-WaxysgRNA-Cas9载体(图1)。
3、CRISPR/Cas9 T0突变体的获得
将该正确的表达载体转化农杆菌EHA105中，以水稻秀水134为受体进行农杆菌转化，经过筛选、分化、再生过程获得T0代转基因植株。将T0代植株分单株种植，单株取叶片提取基因组DNA。针对第4外显子靶位点附近设计引物并进行PCR检测，检测引物为：CRISPRTXT-F:5’-ACCAGTACAAGGACG CTTGG-3’(SEQ ID NO.8)和CRISPR TXT-R:5’-TGCCTGCAGGTAGCATCAAT-3’(SEQ ID NO.9)，将PCR产物进行测序，确定T0代植株发生Waxy纯合突变，突变类型为第849和第850位C突变位T(图2)。
实施例2：直链淀粉含量5％左右的水稻突变体Waxy-m基因克隆
对上述实施例1的纯合突变植株的T1代单株分别取叶片，提取基因组DNA，以Waxy基因全长的特异引物Waxy-F和Waxy-R进行PCR扩增。扩增产物进行测序。测序结果与秀水134野生型Waxy基因(核酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO.4所示)比较，发现该基因的第849和第850位C突变为T的纯合突变，与T0代植株相同，进一步分析，Waxy基因该位点的突变导致其编码的GBSSI蛋白序列与秀水134GBSSI蛋白的第178位氨基酸由苏氨酸突变为异亮氨酸，如图3所示。综合该类纯合突变稻米直链淀粉含量均为5％左右，推测该突变为产生5％左右直链淀粉含量的品质的关键突变位点。
直链淀粉含量5％左右水稻突变体的Waxy基因的核苷酸序列如SEQID NO.1所示，其编码的GBSSI蛋白的氨基酸序列如SEQ ID NO.2所示，将克隆得到的新基因命名为Waxy-m。
本发明鉴定到的Waxy-m基因第849和第850位由野生型C到T的变异，以及因此引起的第178位氨基酸由苏氨酸突变为异亮氨酸的变异均为首次报道。
实施例3去除T-DNA载体的稳定突变体植株的获得
本发明构建的定向编辑Waxy基因的载体，含有T-DNA载体，本发明涉及的T-DNA主要包含了筛选标记HPT基因和Cas9元件，由于潮霉素磷酸转移酶HPT基因和Cas9元件的主要作用是用于转化阳性植株的筛选和完成对目标基因的定点突变，并且这两个基因相对于水稻基因组来说是外源基因，且HPT为抗生素筛选标记，需要剔除，Cas9元件如果保留在植株中有可能继续编辑突变位点，产生其他的Waxy等位基因，而且，T-DNA随机插入还可能造成非预期的基因突变，所以在其完成基因编辑任务后，需要清除。通过农杆菌介导转化秀水134，在转基因过程中，T-DNA序列会随机插入水稻的染色体中，可能以单拷贝或多拷贝插入。由于T-DNA插入位点与其靶位点一般不连锁，因此有望通过转基因植株的后代分离获得不携带T-DNA的植株。为了获得不含上述Cas9元件的植株，本发明人通过对目标基因纯合突变的T1代植株的HYP基因和Cas9元件进行检测，重复3次，筛选不携带HYP基因和Cas9元件的植株，即为剔除HYP基因和Cas9元件的T1代单株。
取上述实施例2的18个单株的基因组DNA，以引物hyg283-F：
TCCGGAAGTGCTTGACATT(SEQ ID NO.10)和hyg283-R：
GTCGTCCATCACAGTTTGC(SEQ ID NO.11)对HPT基因进行PCR扩增；以Cas9元件PCR检测引物，引物序列为：CAS9 TXT-F:5’-GTAAAACGACGGCCAGT-3’(SEQ ID NO.12)和CAS9 TXT-R:5’-TCTAGAGAGGGGCACGACC-3(SEQ ID NO.13)，对Cas9元件进行PCR扩增，筛选出突变类型为纯合突变且HYP基因和Cas9元件检测为阴性的植株。对T1代检测没有HYP基因和Cas9元件的纯合突变体植株进行收种，进行T2代种植，随机筛选部分植株进行基因型检测验证。证明T2代植株中存在的突变类型的纯合突变体，且已经检测不到HYP基因和Cas9元件。
实施例4直链淀粉含量测定
对获得的没有HYP基因和Cas9元件的T2代纯合突变植株，单株收种，随机挑选3株，检测其稻米外观如图4，测定其种子中直链淀粉含量。
应用上海三黍生物科技有限公司提供的直链淀粉含量测定方法，结果见下表：
| 编号 | 生物学重复 | 直链淀粉含量(％) |
| 秀水134(对照) | 1 | 14.53 |
| 秀水134(对照) | 2 | 14.95 |
| 秀水134(对照) | 3 | 15.09 |
| 含有Waxy-m的植株 | 1 | 5.10 |
| 含有Waxy-m的植株 | 2 | 4.96 |
| 含有Waxy-m的植株 | 3 | 5.05 |
发现Waxy-m突变类型植株的直链淀粉含量都在5％左右，属于软米特征，由此证明本发明提供的Waxy基因人工定点突变体能够将水稻直链淀粉含量改变为5％左右。
实施例5：突变体农艺性状调查
将秀水134与实施例3中鉴定到的剔除T-DNA的T2代纯合突变体种子种植于上海市试验基地，秀水134与waxy-m突变体种植小区，每小区100苗，三次重复。对农艺性状进行观察分析，结果表明，waxy-m突变体与秀水134的株型、叶片颜色、株高、分蘖数、穗型、粒长、粒宽等农艺性状进行比较，均无明显差异。因此，编辑获得的新材料保留了秀水134材料的丰产、优质等重要农艺性状。
通过上述具体实施例表明，利用CRISPR/Cas9基因编辑技术，对Waxy基因进行编辑，通过后代的筛选，在T2代就可以获得剔除Cas9元件，特性稳定遗传的新材料，而新材料的基本农艺性状无明显改变。相对于化学诱变、杂交转育等育种方法，基因编辑定向改良分子育种技术具有快速、精准、高效等优点，将会极大提高育种效率，大大加快育种进。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明做出各种改变或者变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
序列表
<110> 上海师范大学
<120> 基于基因编辑技术的GBSSI突变型蛋白及其在植物育种中的应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3480
<212> DNA
<213> 未知(Unknown)
<400> 1
atgtcggctc tcaccacgtc ccagctcgcc acctcggcca ccggcttcgg catcgccgac 60
aggtcggcgc cgtcgtcgct gctccgccac gggttccagg gcctcaagcc ccgcagcccc 120
gccggcggcg acgcgacgtc gctcagcgtg acgaccagcg cgcgcgcgac gcccaagcag 180
cagcggtcgg tgcagcgtgg cagccggagg ttcccctccg tcgtcgtgta cgccaccggc 240
gccggcatga acgtcgtgtt cgtcggcgcc gagatggccc cctggagcaa gaccggcggc 300
ctcggtgacg tcctcggtgg cctcccccct gccatggctg taagcacaca caaacttcga 360
tcgctcgtcg tcgctgaccg tcgtcgtctt caactgttct tgatcatcgc attggatgga 420
tgtgtaatgt tgtgttcttg tgttctttgc aggcgaatgg ccacagggtc atggtgatct 480
ctcctcggta cgaccagtac aaggacgctt gggataccag cgttgtggct gaggtaggag 540
catatgcgtg atcagatcat cacaagatcg attagcttta gatgatttgt tacatttcgc 600
aagattttaa cccaagtttt tgtggtgcaa ttcattgcag atcaaggttg cagacaggta 660
cgagagggtg aggtttttcc attgctacaa gcgtggagtc gaccgtgtgt tcatcgacca 720
tccgtcattc ctggagaagg tggagtcatc attagtttac cttttttgtt tttactgaat 780
tattaacagt gcatttagca gttggactga gcttagcttc cactggtgat ttcaggtttg 840
gggaaagatt ggtgagaaga tctacggacc tgacactgga gttgattaca aagacaacca 900
gatgcgtttc agccttcttt gccaggtcag tgattacttc tatctgatga tggttggaag 960
catcacgagt ttaccatagt atgtatggat tcataactaa ttcgtgtatt gatgctacct 1020
gcaggcagca ctcgaggctc ctaggatcct aaacctcaac aacaacccat acttcaaagg 1080
aacttatggt gagttacaat tgatctcaag atcttataac tttcttcgaa ggaatccatg 1140
atgatcagac taattccttc cggtttgtta ctgacaacag gtgaggatgt tgtgttcgtc 1200
tgcaacgact ggcacactgg cccactggcg agctacctga agaacaacta ccagcccaat 1260
ggcatctaca ggaatgcaaa ggtctatgct tgttcttgcc ataccaactc aaatctgcat 1320
gcacactgca ttctgttcag aaactgactg tctgaatctt tttcactgca ggttgctttc 1380
tgcatccaca acatctccta ccagggccgt ttcgctttcg aggattaccc tgagctgaac 1440
ctctccgaga ggttcaggtc atccttcgat ttcatcgacg ggtatgagta agattctaag 1500
agtaacttac tgtcaattcg ccatatatcg attcaatcca agatcctttt gagctgacaa 1560
ccctgcacta ctgtccatcg ttcaaatccg gttaaatttc aggtatgaca cgccggtgga 1620
gggcaggaag atcaactgga tgaaggccgg aatcctggaa gccgacaggg tgctcaccgt 1680
gagcccgtac tacgccgagg agctcatctc cggcatcgcc aggggatgcg agctcgacaa 1740
catcatgcgg ctcaccggca tcaccggcat cgtcaacggc atggacgtca gcgagtggga 1800
tcctagcaag gacaagtaca tcaccgccaa gtacgacgca accacggtaa gaacgaatgc 1860
attcttcaca agatatgcaa tctgaatttt ctttgaaaaa gaaattatca tctgtcactt 1920
cttgattgat tctgacaagg caagaatgag tgacaaattt caggcaatcg aggcgaaggc 1980
gctgaacaag gaggcgttgc aggcggaggc gggtcttccg gtcgacagga aaatcccact 2040
gatcgcgttc atcggcaggc tggaggaaca gaagggccct gacgtcatgg ccgccgccat 2100
cccggagctc atgcaggagg acgtccagat cgttcttctg gtataatata atacactaca 2160
agacacactt gcacgatatg ccaaaaattc agaacaaatt cagtggcaaa aaaaaaactc 2220
gaatattagg gaaggaccta ataatatcaa ataattagaa ggggtgaggc tttgaaccca 2280
gatcgtctag tccaccacct tgtggagtta gccggaagac ctctgagcat ttctcaattc 2340
agtggcaaat gatgtgtata attttgatcc gtgtgtgttt cagggtactg gaaagaagaa 2400
gttcgagaag ctgctcaaga gcatggagga gaagtatccg ggcaaggtga gggccgtggt 2460
gaagttcaac gcgccgcttg ctcatctcat catggccgga gccgacgtgc tcgccgtccc 2520
cagccgcttc gagccctgtg gactcatcca gctgcagggg atgagatacg gaacggtata 2580
caatttccat ctatcaattc gattgttcga tttcatcttt gtgcaatgca atgcaattgc 2640
aaatgcaaat gcatgatgat tttccttgtt gatttctcca gccctgtgct tgcgcgtcca 2700
ccggtgggct cgtggacacg gtcatcgaag gcaagactgg tttccacatg ggccgtctca 2760
gcgtcgacgt aagcctatac atttacataa caatcagata tgacacatcc taataccgat 2820
aagtcggtac actactacac atttacatgg ttgctggtta tatggttttt ttggcagtgc 2880
aaggtggtgg agccaagcga cgtgaagaag gtggcggcca ccctgaagcg cgccatcaag 2940
gtcgtcggca cgccggcgta cgaggagatg gtcaggaact gcatgaacca ggacctctcc 3000
tggaaggtat aaattacgaa acaaatttaa cccaaacata tactatatac tccctccgct 3060
tctaaatatt caacgccgtt gtctttttta aatatgtttg accattcgtc ttattaaaaa 3120
aattaaataa ttataaattc ttttcctatc atttgattca ttgttaaata tacttatatg 3180
tatacatata gttttacata tttcataaaa ttttttgaac aagacgaacg gtcaaacatg 3240
tgctaaaaag ttaacggtgt cgaatattca gaaacggagg gagtataaac gtcttgttca 3300
gaagttcaga gattcacctg tctgatgctg atgatgatta attgtttgca acatggattt 3360
caggggcctg cgaagaactg ggagaatgtg ctcctgggcc tgggcgtcgc cggcagcgcg 3420
ccggggatcg aaggcgacga gatcgcgccg ctcgccaagg agaacgtggc tgctccttga 3480
<210> 2
<211> 609
<212> PRT
<213> 未知(Unknown)
<400> 2
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser
85 90 95
Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110
Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln
115 120 125
Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala
130 135 140
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val
145 150 155 160
Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly
165 170 175
Lys Ile Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190
Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala
195 200 205
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr
210 215 220
Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240
Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn
245 250 255
Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
260 265 270
Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285
Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys
290 295 300
Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly
325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val
340 345 350
Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile
355 360 365
Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn
370 375 380
Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile
385 390 395 400
Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile
420 425 430
Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser
435 440 445
Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn
450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val
465 470 475 480
Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
485 490 495
Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr
500 505 510
Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
515 520 525
Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu
530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val
545 550 555 560
Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
565 570 575
Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly
580 585 590
Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605
Pro
<210> 3
<211> 3480
<212> DNA
<213> 未知(Unknown)
<400> 3
atgtcggctc tcaccacgtc ccagctcgcc acctcggcca ccggcttcgg catcgccgac 60
aggtcggcgc cgtcgtcgct gctccgccac gggttccagg gcctcaagcc ccgcagcccc 120
gccggcggcg acgcgacgtc gctcagcgtg acgaccagcg cgcgcgcgac gcccaagcag 180
cagcggtcgg tgcagcgtgg cagccggagg ttcccctccg tcgtcgtgta cgccaccggc 240
gccggcatga acgtcgtgtt cgtcggcgcc gagatggccc cctggagcaa gaccggcggc 300
ctcggtgacg tcctcggtgg cctcccccct gccatggctg taagcacaca caaacttcga 360
tcgctcgtcg tcgctgaccg tcgtcgtctt caactgttct tgatcatcgc attggatgga 420
tgtgtaatgt tgtgttcttg tgttctttgc aggcgaatgg ccacagggtc atggtgatct 480
ctcctcggta cgaccagtac aaggacgctt gggataccag cgttgtggct gaggtaggag 540
catatgcgtg atcagatcat cacaagatcg attagcttta gatgatttgt tacatttcgc 600
aagattttaa cccaagtttt tgtggtgcaa ttcattgcag atcaaggttg cagacaggta 660
cgagagggtg aggtttttcc attgctacaa gcgtggagtc gaccgtgtgt tcatcgacca 720
tccgtcattc ctggagaagg tggagtcatc attagtttac cttttttgtt tttactgaat 780
tattaacagt gcatttagca gttggactga gcttagcttc cactggtgat ttcaggtttg 840
gggaaagacc ggtgagaaga tctacggacc tgacactgga gttgattaca aagacaacca 900
gatgcgtttc agccttcttt gccaggtcag tgattacttc tatctgatga tggttggaag 960
catcacgagt ttaccatagt atgtatggat tcataactaa ttcgtgtatt gatgctacct 1020
gcaggcagca ctcgaggctc ctaggatcct aaacctcaac aacaacccat acttcaaagg 1080
aacttatggt gagttacaat tgatctcaag atcttataac tttcttcgaa ggaatccatg 1140
atgatcagac taattccttc cggtttgtta ctgacaacag gtgaggatgt tgtgttcgtc 1200
tgcaacgact ggcacactgg cccactggcg agctacctga agaacaacta ccagcccaat 1260
ggcatctaca ggaatgcaaa ggtctatgct tgttcttgcc ataccaactc aaatctgcat 1320
gcacactgca ttctgttcag aaactgactg tctgaatctt tttcactgca ggttgctttc 1380
tgcatccaca acatctccta ccagggccgt ttcgctttcg aggattaccc tgagctgaac 1440
ctctccgaga ggttcaggtc atccttcgat ttcatcgacg ggtatgagta agattctaag 1500
agtaacttac tgtcaattcg ccatatatcg attcaatcca agatcctttt gagctgacaa 1560
ccctgcacta ctgtccatcg ttcaaatccg gttaaatttc aggtatgaca cgccggtgga 1620
gggcaggaag atcaactgga tgaaggccgg aatcctggaa gccgacaggg tgctcaccgt 1680
gagcccgtac tacgccgagg agctcatctc cggcatcgcc aggggatgcg agctcgacaa 1740
catcatgcgg ctcaccggca tcaccggcat cgtcaacggc atggacgtca gcgagtggga 1800
tcctagcaag gacaagtaca tcaccgccaa gtacgacgca accacggtaa gaacgaatgc 1860
attcttcaca agatatgcaa tctgaatttt ctttgaaaaa gaaattatca tctgtcactt 1920
cttgattgat tctgacaagg caagaatgag tgacaaattt caggcaatcg aggcgaaggc 1980
gctgaacaag gaggcgttgc aggcggaggc gggtcttccg gtcgacagga aaatcccact 2040
gatcgcgttc atcggcaggc tggaggaaca gaagggccct gacgtcatgg ccgccgccat 2100
cccggagctc atgcaggagg acgtccagat cgttcttctg gtataatata atacactaca 2160
agacacactt gcacgatatg ccaaaaattc agaacaaatt cagtggcaaa aaaaaaactc 2220
gaatattagg gaaggaccta ataatatcaa ataattagaa ggggtgaggc tttgaaccca 2280
gatcgtctag tccaccacct tgtggagtta gccggaagac ctctgagcat ttctcaattc 2340
agtggcaaat gatgtgtata attttgatcc gtgtgtgttt cagggtactg gaaagaagaa 2400
gttcgagaag ctgctcaaga gcatggagga gaagtatccg ggcaaggtga gggccgtggt 2460
gaagttcaac gcgccgcttg ctcatctcat catggccgga gccgacgtgc tcgccgtccc 2520
cagccgcttc gagccctgtg gactcatcca gctgcagggg atgagatacg gaacggtata 2580
caatttccat ctatcaattc gattgttcga tttcatcttt gtgcaatgca atgcaattgc 2640
aaatgcaaat gcatgatgat tttccttgtt gatttctcca gccctgtgct tgcgcgtcca 2700
ccggtgggct cgtggacacg gtcatcgaag gcaagactgg tttccacatg ggccgtctca 2760
gcgtcgacgt aagcctatac atttacataa caatcagata tgacacatcc taataccgat 2820
aagtcggtac actactacac atttacatgg ttgctggtta tatggttttt ttggcagtgc 2880
aaggtggtgg agccaagcga cgtgaagaag gtggcggcca ccctgaagcg cgccatcaag 2940
gtcgtcggca cgccggcgta cgaggagatg gtcaggaact gcatgaacca ggacctctcc 3000
tggaaggtat aaattacgaa acaaatttaa cccaaacata tactatatac tccctccgct 3060
tctaaatatt caacgccgtt gtctttttta aatatgtttg accattcgtc ttattaaaaa 3120
aattaaataa ttataaattc ttttcctatc atttgattca ttgttaaata tacttatatg 3180
tatacatata gttttacata tttcataaaa ttttttgaac aagacgaacg gtcaaacatg 3240
tgctaaaaag ttaacggtgt cgaatattca gaaacggagg gagtataaac gtcttgttca 3300
gaagttcaga gattcacctg tctgatgctg atgatgatta attgtttgca acatggattt 3360
caggggcctg cgaagaactg ggagaatgtg ctcctgggcc tgggcgtcgc cggcagcgcg 3420
ccggggatcg aaggcgacga gatcgcgccg ctcgccaagg agaacgtggc tgctccttga 3480
<210> 4
<211> 609
<212> PRT
<213> 未知(Unknown)
<400> 4
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser
85 90 95
Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110
Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln
115 120 125
Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala
130 135 140
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val
145 150 155 160
Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly
165 170 175
Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190
Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala
195 200 205
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr
210 215 220
Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240
Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn
245 250 255
Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
260 265 270
Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285
Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys
290 295 300
Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly
325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val
340 345 350
Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile
355 360 365
Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn
370 375 380
Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile
385 390 395 400
Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile
420 425 430
Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser
435 440 445
Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn
450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val
465 470 475 480
Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
485 490 495
Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr
500 505 510
Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
515 520 525
Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu
530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val
545 550 555 560
Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
565 570 575
Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly
580 585 590
Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605
Pro
<210> 5
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 5
aagaccggtg agaagatcta 20
<210> 6
<211> 25
<212> DNA
<213> 未知(Unknown)
<400> 6
tgtgtgagac cggtgagaag atcta 25
<210> 7
<211> 25
<212> DNA
<213> 未知(Unknown)
<400> 7
aaactagatc ttctcaccgg tctca 25
<210> 8
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 8
accagtacaa ggacgcttgg 20
<210> 9
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 9
tgcctgcagg tagcatcaat 20
<210> 10
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 10
tccggaagtg cttgacatt 19
<210> 11
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 11
gtcgtccatc acagtttgc 19
<210> 12
<211> 17
<212> DNA
<213> 未知(Unknown)
<400> 12
gtaaaacgac ggccagt 17
<210> 13
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 13
tctagagagg ggcacgacc 19
Claims (10)
1.一种水稻GBSSI突变型蛋白，其特征在于，所述GBSSI突变型蛋白的氨基酸序列存在以下突变：其对应于水稻GBSSI氨基酸序列的第178位氨基酸发生突变。
2.根据权利要求1所述的水稻GBSSI突变型蛋白，其特征在于，包括：
(a)其氨基酸序列如SEQ ID NO.2所示；或
(b)在(a)中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具有直链淀粉合成酶活性的由(a)衍生的蛋白质。
3.一种编码权利要求1或2所述突变型蛋白的基因。
4.根据权利要求3所述的基因，其特征在于：
a)其核苷酸序列如SEQ ID NO.1所示；或
b)在严格条件下与a)限定的核酸序列杂交且编码具有直链淀粉合成酶活性蛋白质的核酸序列。
5.表达盒、重组载体或细胞，其特征在于，所述表达盒、重组载体或细胞含有权利要求3或4所述基因。
6.权利要求1或2所述水稻GBSSI突变型蛋白在水稻育种中的应用。
7.获得低直链淀粉含量的水稻的方法，其特征在于，包括如下步骤：
1)使水稻植株包含权利要求3或4所述的基因；或
2)使水稻植株表达权利要求1或2所述的水稻GBSSI突变型蛋白。
8.一种利用基因编辑技术低直链淀粉含量水稻的育种方法，其特征在于，包括以下步骤：
1)设计Waxy基因定点编辑的靶位点：基因编辑的靶位点核苷酸序列如SEQ ID NO.5所示；
2)构建含有目的片段的CRISPR/Cas9基因编辑载体：
A)靶点接头制备：采用接头引物用双蒸水溶解成母液，母液稀释后90℃30s(second，秒)，移至室温冷却完成退火，即获得靶点接头；所述引物为Waxy TXT-F和Waxy TXT-R，序列如SEQ ID NO.8和SEQ ID NO.9所示；
B)将退火获得的靶点片段连接到CRISPR/Cas9表达载体上获得连接产物；
C)将步骤B)的连接产物进行热激法转化大肠杆菌获得重组菌，提取通过验证的含目的条带的菌液的阳性质粒；
3)将阳性质粒转化农杆菌EHA105，获得T0代转基因植株，以引物Waxy TXT-F和WaxyTXT-R对T0代转基因植株进行扩增并测序鉴定获得具备权利要求1或2所述突变型蛋白的植株。
9.根据权利要求8所述的育种方法，其特征在于，所述育种方法还包括将具有突变型蛋白的T0代转基因植株的含有目标等位基因纯合突变的T1代植株的T-DNA载体的剔除，所述T-DNA载体包括筛选标记HPT基因和Cas9元件。
10.根据权利要求9所述的育种方法，其特征在于，所述T-DNA载体的剔除通过对含有目标等位基因纯合突变的T1代植株的HPT基因和Cas9元件同时检测，重复多次，筛选得到不携带这两个基因的T1代单株即为目标植株。
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| CN111197034A (zh) * | 2020-01-08 | 2020-05-26 | 江苏省农业科学院 | 基于基因编辑技术的Wx突变型蛋白及其基因在植物育种中的应用 |
| CN113564197A (zh) * | 2021-07-08 | 2021-10-29 | 上海师范大学 | 一种CRISPR/Cas9介导的植物多基因编辑载体的构建方法和应用 |
| CN114085853A (zh) * | 2021-11-25 | 2022-02-25 | 湖南省核农学与航天育种研究所 | Waxy突变体及其筛选方法和用途 |
| CN115197952A (zh) * | 2022-05-17 | 2022-10-18 | 重庆市农业科学院 | 水稻蜡质基因Wx的突变基因及其应用 |
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| CN114085853A (zh) * | 2021-11-25 | 2022-02-25 | 湖南省核农学与航天育种研究所 | Waxy突变体及其筛选方法和用途 |
| CN114085853B (zh) * | 2021-11-25 | 2024-05-17 | 湖南省核农学与航天育种研究所 | Waxy突变体及其筛选方法和用途 |
| CN115197952A (zh) * | 2022-05-17 | 2022-10-18 | 重庆市农业科学院 | 水稻蜡质基因Wx的突变基因及其应用 |
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