CN114540369B - OsBEE1基因在提高水稻产量中的应用 - Google Patents

OsBEE1基因在提高水稻产量中的应用 Download PDF

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CN114540369B
CN114540369B CN202210192351.1A CN202210192351A CN114540369B CN 114540369 B CN114540369 B CN 114540369B CN 202210192351 A CN202210192351 A CN 202210192351A CN 114540369 B CN114540369 B CN 114540369B
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陈震
韩迎春
李俊周
赵全志
杜彦修
孙红正
张静
彭廷
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Abstract

本发明属于植物基因工程技术领域，具体涉及OsBEE1基因在提高水稻产量中的应用。本发明根据OsBEE1基因的编码序列，依据CRISPR/Cas9技术原理进行定向编辑，选择OsBEE1基因的突变靶点，构建CRISPR/Cas9‑gRNA表达载体。利用农杆菌介导法导入水稻，以除草剂抗性标记筛选获得阳性转基因植株，利用测序法分析鉴定突变单株，获得具有育种应用价值的水稻OsBEE1突变新种质。与野生型相比，突变后代的籽粒千粒重显著增加，增幅达5.12～7.08%，表明OsBEE1基因与控制水稻粒重有关，可应用于水稻高产育种。

Description

OsBEE1基因在提高水稻产量中的应用
技术领域
本发明属于植物基因工程技术领域，具体涉及控制水稻粒重的OsBEE1基因、其CRISPR/Cas9载体及应用。
背景技术
水稻(Oryza sativa L.)为全球半数以上人口提供食物来源，也是我国最重要的粮食作物之一，约占我国粮食总产量的三分之一。水稻的稳产和增产对保障我国粮食安全具有重要的战略意义。水稻产量主要由穗数、每穗粒数和千粒重决定，而粒重是决定水稻产量的三要素中遗传力最高的因素。粒重主要由粒长、粒宽、粒厚和籽粒充实度决定(Huanget al.,Genetic bases of rice grain shape:so many genes,so little known.TrendsPlant Sci,2013,18,218-226.)。目前许多粒重和粒型相关性状基因相继被克隆，如：GS3、GS5、GW2、GW5、GW7和GW8。其中GS3是控制水稻粒重和粒长的主效QTL(Fan et al.,GS3,amajor QTL for grain length and weight and minor QTL for grain width andthickness in rice,encodes a putative transmembrane protein.Theor Appl Genet,2006,112,1164-1171.)，GS5是控制水稻粒宽、充实度和千粒重的数量性状基因(Li etal.,Natural variation in GS5 plays an important role in regulating grain sizeand yield in rice.Nat Genet,2011,43,1266-1269.)；GW2负调控籽粒粒宽和粒重(Songet al.,A QTL for rice grain width and weight encodes a previously unknownRING-type E3 ubiquitin ligase.Nat Genet,2007,39,623-630.)，GW5可通过影响泛素蛋白酶体途径和油菜素内酯信号途径控制籽粒粒宽和粒重(Weng et al.,Isolation andinitial characterization of GW5,a major QTL associated with rice grain widthand weight.Cell Res,2008,18,1199-1209.；Liu et al.,GW5 acts in thebrassinosteroidsignalling pathway to regulate grain width and weight inrice.Nat Plants,2017,3,17043.)，GW8编码转录因子OsSPL16，能够直接与GW7启动子结合并抑制其表达，从而控制籽粒大小和品质(Wang et al.,Control of grain size,shapeand quality by OsSPL16 in rice.Nat Genet,2012,44,950-954.；Wang et al.,TheOsSPL16-GW7 regulatory module determines grain shape and simultaneouslyimproves rice yield and grain quality.Nat Genet,2015,47,949-954.)。
BEE(BR enhanced expression)基因属于bHLH转录因子家族成员，其表达受BRs信号调控。bHLH转录因子是水稻、玉米、拟南芥等高等植物中最大转录因子家族之一(Carretero-Paulet et al.,Genome-wide classification and evolutionary analysisof the bHLH family of transcription factors in Arabidopsis,poplar,rice,moss,and algae.Plant Physiol,2010,153,1398-1412.)。bHLH转录因子的命名是基于其含有的约60个氨基酸的两个功能不同的结构域：位于N端碱性区域(Basic region)，一般由15个氨基酸组成，是DNA的结合区域；位于C端的螺旋-环-螺旋区域(Helix-Loop-Helix)，约由45个氨基酸组成，含有两个被不同长度的环分开的亲水亲脂的、能与E-box/G-box顺式作用元件特异性结合α-螺旋，调控下游基因的表达(Li et al.,Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis.PlantPhysiol,2006,141,1167-1184.；Qian et al.,Regulatory mechanisms of bHLHtranscription factors in plant adaptive responses to various abioticstresses.Front Plant Sci,2021,12,677611.)。基于全基因组测序和生物信息学预测，水稻中发现167个bHLH转录因子，在形态建成(Zhang et al.,Antagonistic HLH/bHLHtranscription factors mediate brassinosteroid regulation of cell elongationand plant development in rice and Arabidopsis.Plant Cell,2009,21,3767-3780.；Dong et al.,Genome-wide association studies reveal that members of bHLHsubfamily 16 share a conserved function in regulating flag leaf angle in rice(Oryza sativa).Plos Genet,2018,14,e1007323.)、生长发育(Dinget al.,Atranscription factor with a bHLH domain regulates root hair development inrice.Cell Res,2009,19,1309-1311.；Heang et al.,2012；Fuet al.,The rice basicHelix-Loop-Helix transcription factor TDR INTERACTING PROTEIN2 is a centralswitch in early anther development.Plant Cell,2014,26,1512-1524.)、抗逆反应(Seo et al.,OsbHLH148,a basic helix-loop-helix protein,interacts with OsJAZproteins in a jasmonate signaling pathway leading to drought tolerance inrice.Plant J,2011,65,907-921.；Chihiro Yamamura et al.,2015Diterpenoidphytoalexin factor,a bHLHtranscription factor,plays a central role in thebiosynthesis of diterpenoid phytoalexins in rice.Plant J,2015,84.)等方面都有着重要作用。在水稻粒型和粒重调控中也发现有bHLH转录因子的参与，如OsbHLH107参与调控水稻籽粒粒长(Yang et al.,Overexpression of OsbHLH107,a member of the basichelix-loop-helix transcription factor family,enhances grain size in rice(Oryza sativa L.).Rice,2018,11,41.)，OsPGLl和OsPGL2形成二聚体抑制APG的表达从而调控籽粒大小和粒重(Heang et al.,Antagonistic actions of HLH/bHLH proteins areinvolved in grain length and weight in rice.PloS one,2012,7,e31325.；Heangetal.,An atypical bHLH protein encoded by POSITIVE REGULATOR OF GRAINLENGTH 2 is involved in controlling grain length and weight of rice throughinteraction with a typical bHLH protein APG.Breed Sci,2012,62,133-141.)，OsPIL15可直接调控靶基因OsPUP7的表达，影响细胞分裂素转运进而影响细胞分裂，从而负向调控籽粒大小(Ji et al.The basic helix-loop-helix transcription factor,OsPIL15,regulates grain size via directly targeting a purine permease geneOsPUP7 in rice.Plant Biotechnol J,2019,17,1527-1537.)。
基因编辑技术能够实现对特定目标基因的基因组序列进行定点修饰，包括对特定DNA序列的插入、替换和缺失等，产生不同类型的等位基因突变。CRISPR/Cas9(Clusteredregularly interspaced short palindromic repeats/CRISPR-associated nuclease 9,Cas9)基因组定向编辑方法是近几年应用比较广泛的一种准确、便捷、高效率的生物基因组定向编辑技术。CRISPR/Cas9技术是利用核酸酶Cas9蛋白与单导向RNA(Single guide RNA,sgRNA)形成复合体使特定的靶基因定点突变；sgRNA通过碱基互补配对决定靶序列特异性，Cas9蛋白作为核酸酶切割与sgRNA上的间隔序列(Spacers)互补的基因组DNA，造成双链DNA损伤，随后通过体内的NHEJ(Non-homologous end joining)修复机制引入基因突变(Wiedenheft et al.,RNA-guided genetic silencing systems in bacteria andarchaea.Nature,2012,482,331-338.)，通常在PAM位点的上游3bp处实现插入或缺失。随着CRISPR/Cas9系统建立与应用，目前在拟南芥、水稻、小麦、玉米、烟草等植物中均实现了基因组的定向编辑，控制水稻粒长的OsbHLH107和OsPIL15基因经敲除后，转基因植株的籽粒粒型、粒重发生改变。
发明内容
对于水稻转录因子OsBEE1基因，本发明发现敲除水稻中的该基因，可以提高水稻的粒重，增加粒宽。
具体的，本发明提供OsBEE1基因在提高水稻产量中的应用，敲除水稻的OsBEE1基因增加粒宽、提高水稻的粒重；所述OsBEE1基因的核苷酸编码序列如SEQ ID NO:1所示，该基因编码的氨基酸序列如SEQ ID NO:2所示。OsBEE1基因全长序列如SEQ ID NO:5所示。
本发明还提供了敲除OsBEE1基因的试剂盒在提高水稻产量中的应用，具体表现为提高增加粒宽、水稻的粒重。
上述敲除OsBEE1基因的试剂中包含以OsBEE1基因为靶点的CRISPR/Cas9-gRNA表达载体。
上述gRNA的靶点序列为5′-ACACCACTCTCTCCTCCTCAAGG-3′，PAM序列为3′端AGG。gRNA寡核苷酸链的上游引物为BEE1_gRNA_F:5’-GGCGACACCACTCTCTCCTCCTCA-3’，如SEQ IDNO:3所示，下游引物为BEE1_sRNA_R:5’-AAACTGAGGAGGAGAGAGTGGTGT-3’，如SEQ ID NO:4所示。
为了实现上述目的，本发明所采用的技术方案是：
一种基于CRISPR/Cas9技术制备水稻OsBEE1突变体的方法，包括如下步骤：
(1)gRNA靶点序列的选择：靶点序列为5′-ACACCACTCTCTCCTCCTCAAGG-3′，所述PAM序列为3′端AGG；
(2)gRNA寡核苷酸链上下游引物的设计：
上游引物为BEE1_gRNA_F:5′-GGCGACACCACTCTCTCCTCCTCA-3′，
下游引物为BEE1_gRNA_R:5′-AAACTGAGGAGGAGAGAGTGGTGT-3′；
(3)gRNA表达载体构建：将gRNA寡核苷酸链上下游引物混合、退火，得寡核苷酸双链DNA；用内切酶BsaI酶切CRISPR/Cas9质粒(如pBUN411质粒)，得线性质粒；用T4连接酶将所述线性质粒和寡核苷酸双链DNA连接，得连接产物；将连接产物转化、筛选、验证，即得CRISPR/Cas9-gRNA表达载体；
其中，所述退火为在65℃退火5min；所述酶切的体系为：pBUN411质粒1μg，10×CutSmart Buffer 5μL，BsaI1μL，加ddH2O至50μL；所述酶切的条件为：37℃酶切3h；T4连接酶连接的体系为：线性质粒3μL，寡核苷酸双链DNA 1μL，10×Buffer 1μL，T4 DNA Ligase0.5μL，加ddH2O至10μL。T4 DNA Ligase连接的条件为：25℃连接1h。
将连接产物转化、筛选、验证的具体操作为：将连接产物用热激法转化大肠杆菌DH5α感受态细胞，菌液涂布于含50mg/L卡那霉素的LB培养基平板，过夜培养后挑取单克隆摇菌扩繁；进行菌落PCR验证。所述LB培养基的配方为(1L)：胰蛋白胨10g，酵母提取物5g，氯化钠10g，蒸馏水定容至1L；固体LB培养基加入琼脂粉10g/L。
(4)将CRISPR/Cas9-gRNA表达载体导入农杆菌中，得CRISPR/Cas9-gRNA农杆菌；用CRISPR/Cas9-gRNA农杆菌侵染水稻愈伤组织；其中，采用热激法将CRISPR/Cas9-gRNA表达载体导入土壤农杆菌EHA105。
(5)将步骤(4)获得的愈伤组织诱导得到再生苗，筛选得到转基因阳性植株，鉴定即得水稻OsBEE1突变体。
步骤(5)中，在除草剂的条件下筛选得到再生苗；将所述转基因阳性植株的PCR扩增产物测序，确定是纯合/杂合突变体，或者是未发生突变单株。
其中，转基因阳性植株采用抗除草剂基因引物进行筛选，筛选出扩增产物为412bp的植株，进行进一步鉴定；所用引物为：Bar_F：5’-AAGCACGGTCAACTTCCGTA-3’；Bar_R：5’-GAAGTCCAGCTGCCAGAAAC-3’。突变鉴定采用gRNA靶点序列两侧引物进行，两侧引物为：5’-BEE1_cas9_F：TGCTCTACTCTTTAGCCTCTG-3’，BEE1_cas9_R：5’-CTGCCTTCCTCTTCCTGT-3’；所述鉴定包括：A：以转基因阳性植株DNA为模板，BEE1_cas9_F和BEE1_cas9_R为检测引物，进行PCR扩增；B：PCR扩增体系为：2×Taq Mix 10μL，BEE1_cas9_F 0.5μL，BEE1_cas9_R0.5μL，DNA模板1μL，加ddH2O至20μL。PCR扩增条件为：预变性94℃3min，变性94℃30s，退火57℃30s，延伸72℃30s，终延伸72℃5min，其中变性，退火和延伸为30个循环。C：中采用1％琼脂糖凝胶进行电泳检测，扩增产物片段大小为362bp。
本发明还提供由上述制备水稻OsBEE1突变体的方法制得的水稻OsBEE1突变体在提高水稻产量中的应用。具体的，选择所述水稻OsBEE1突变体中的纯合突变体用于水提高水稻产量(如粒重增加、粒宽增加)。如，选择T0代双等位突变体自交得T1代，从包含T1代的后继世代中选择纯合突变体用于提高水稻产量。
本发明的有益效果为：
本发明依据CRISPR/Cas9技术原理对水稻转录因子OsBEE1进行定向编辑。选择OsBEE1突变靶点，构建CRISPR/Cas9-gRNA(pBUN411-gRNA)表达载体，利用农杆菌介导法导入水稻(粳稻品种日本晴)，以除草剂抗性标记筛选获得阳性转基因植株，利用测序法鉴定纯合突变单株。通过该方法获得了具有应用价值的水稻OsBEE1突变体新种质，与野生型水稻相比，制备的OsBEE1突变体籽粒粒重、粒宽显著增加。
本发明针对当前人口不断增长和粮食需求不断增加的现状，探讨了转录因子OsBEE1在水稻品种改良中的功能，为通过调控(如敲除)OsBEE1基因表达来控制水稻粒重提供了试验依据。本发明基于CRISPR/Cas9技术对水稻OsBEE1突变体创制的成功实施，为快速创建生产上具有应用价值的水稻新品系提供一种简单有效的技术手段，在提高水稻粒重以及增加作物产量上具有潜在的应用价值，对水稻高产和稳产育种具有重要的实践意义。
附图说明
图1为gRNA靶位点及CRISPR/Cas9-gRNA表达载体的组装示意图；
图2为T0代突变体OsBEE1基因扩增产物的琼脂糖凝胶电泳图；
图3为不同OsBEE1突变体突变序列与野生型日本晴序列分析比对图；
图4为OsBEE1突变体与野生型日本晴籽粒粒重差异分析图；
图5为OsBEE1突变体与野生型日本晴籽粒粒宽比较图。
具体实施方式
下面通过具体实施方式对本发明进行更加详细的说明，以便于对本发明技术方案的理解，但并不用于对本发明保护范围的限制。
以下实施例中的引物合成及DNA测序由生工生物工程(上海)股份有限公司完成。
实施例1
1、OsBEE1基因gRNA靶点序列的选择及gRNA寡核苷酸链的上下游引物的设计根据水稻OsBEE1基因的编码区序列设计gRNA靶点序列为5′-ACACCACTCTCTCCTCCTCAAGG-3′，PAM序列为3′端AGG，Cas9蛋白将在AGG序列上游第3～4bp处剪切DNA形成平滑末端。PAM序列及酶切位点如图1所示。
根据gRNA靶点序列设计gRNA寡核苷酸链的上、下游引物，引物序列见表1中BEE1_gRNA_F和BEE1_gRNA_R。
表1引物序列表
引物名称 引物序列(5'～3')
BEE1_gRNA_F GGCGACACCACTCTCTCCTCCTCA
BEE1_gRNA_R AAACTGAGGAGGAGAGAGTGGTGT
pBUN411_VF CCATGAAGCCTTTCAGGACATGTA
pBUN411_VR ACGCTGCAAACATGAGACGGAGAA
Bar_F AAGCACGGTCAACTTCCGTA
Bar_R GAAGTCCAGCTGCCAGAAAC
BEE1_cas9_F TGCTCTACTCTTTAGCCTCTG
BEE1_cas9_R CTGCCTTCCTCTTCCTGT
2、pBUN411-gRNA(CRISPR/Cas9-gRNA)表达载体的构建
1)取等量的gRNA寡核苷酸链上、下游引物(终浓度10μM)混合，经65℃退火5min后逐渐冷却至室温形成互补双链DNA，用于后续载体的构建；
2)使用限制性内切酶BsaI酶切CRISPR/Cas9载体pBUN411(CRISPR/Cas9载体pBUN411由中国农业大学陈其军教授惠赠(Xing et al.A CRISPR/Cas9 toolkit formultiplex genome editing in plants.BMC Plant Biol,2014,14,327.)，使其线性化，50μL酶切体系如下：pBUN411质粒1μg，10×CutSmart Buffer 5μL，BsaI1μL，加ddH2O至50μL；37℃酶切3h；纯化后加入如下连接体系：线性质粒3μL，寡核苷酸双链DNA 1μL，10×Buffer1μL，T4 DNA Ligase 0.5μL，加ddH2O至10μL；连接的条件为：25℃连接1h。
本发明所用的CRISPR/Cas9载体pBUN411也可替换为其他市售CRISPR/Cas9载体。
3)连接产物用热激法转化大肠杆菌DH5α感受态细胞，菌液涂布于含50mg/L 的卡那霉素LB培养基平板，过夜培养后挑取单克隆摇菌扩繁。使用引物pBUN411_VF和pBUN411_VR(引物序列见表1)进行菌落PCR验证。
PCR体系为：2×Taq Mix 10μL，pBUN411_VF(10μM)0.5μL，pBUN411_VR(10μM)0.5μL，单克隆模板1μL，加ddH2O至20μL。PCR条件为：预变性94℃3min，变性94℃30s，退火55℃30s，延伸72℃30s，终延伸72℃5min，其中变性，退火和延伸为30个循环。含未连接有靶位点序列的pBUN411空载的菌落，扩增片段长度为1538bp，含连接有靶位点序列的载体菌落，扩增片段长度应为336bp，选取PCR片段大小正确的菌落，提取质粒，表明pBUN411-gRNA表达载体构建成功。
所述靶位点序列由OsU3基因启动子驱动，编码Cas9蛋白的基因由玉米泛素基因(Ubi)启动子驱动(如图1：CRISPR/Cas9-gRNA表达载体LB和RB之间线性结构所示)。
实施例2农杆菌介导水稻愈伤遗传转化和阳性转基因植株检测采用热激法将CRISPR/Cas9-gRNA表达载体导入农杆菌EHA105感受态细胞，使用含有CRISPR/Cas9-gRNA质粒的农杆菌侵染水稻品种日本晴(Oryza sativa ssp.Japonica cv.Nipponbare)的愈伤组织，参照Nishimura等(Nishimura et al.A protocol for Agrobacterium-mediatedtransformation in rice.Nat Protoc,2006,1,2796-2802)报道方法进行水稻转基因，用除草剂(Basta)筛选获得再生苗。采用CTAB法提取转基因植株基因组DNA，使用抗除草剂基因(bar)引物Bar_F和Bar_R(引物序列见表1)进行转基因植株阳性筛选。
PCR体系为：2×Taq Mix 10μL，Bar_F(10μM)0.5μL，Bar_R(10μM)0.5μL，DNA模板1μL，加ddH2O至20μL。PCR条件为：预变性94℃3min，变性94℃30s，退火52℃30s，延伸72℃30s，终延伸72℃5min，其中变性，退火和延伸为30个循环。PCR扩增产物长度为412bp，即为阳性转基因植株。
实施例3T0代突变体筛选鉴定
1)为了检测获得T0代阳性转基因植株靶位点的突变情况，根据水稻转录因子OsBEE1基因全长序列(如SEQ ID NO:5所示)，在靶位点两侧设计引物BEE1_cas9_F和BEE1_cas9_R(引物序列见表1)，以转基因阳性单株的基因组DNA为模板扩增含靶位点的序列，20μL扩增体系如下：2×Taq Mix 10μL，BEE1_cas9_F(10μM)0.5μL，BEE1_cas9_R(10μM)0.5μL，DNA模板1μL，加ddH2O至20μL。PCR条件：预变性94℃3min，变性94℃30s，退火57℃30s，延伸72℃30s，终延伸72℃5min，其中变性，退火和延伸为30个循环。
将一部分T0代阳性转基因植株的PCR扩增产物使用琼脂糖凝胶电泳分离，结果如图2所示，扩增片段大小为362bp左右。将突变单株的PCR产物送测序，测序结果与野生型日本晴序列进行比对分析突变株基因型，综合分析测序结果得到株系突变情况。本实施例共得到5株纯合突变和7株杂合突变体，包含3种不同突变基因型，3种不同突变基因型如图3所示。
实施例4T1代OsBEE1纯合突变体籽粒表型分析
水稻转录因子OBEE1基因的核苷酸编码序列如SEQ ID NO:1所示，该基因编码的氨基酸序列如SEQ ID NO:2所示。
将实施例3中获得T0代突变体经1代自交后选择得到T1代OsBEE1纯合突变体，选取突变株系BEE1KO-3和BEE1KO-6进行籽粒表型分析。
突变体BEE1KO-3中，突变的OsBEE1的编码序列如SEQ ID NO:6所示；氨基酸序列如SEQ ID NO:7所示。
突变体BEE1KO-6中，突变的OsBEE1的编码序列如SEQ ID NO:8所示；氨基酸序列如SEQ ID NO:9所示。
选取上述2个纯合突变株系BEE1KO-3和BEE1KO-6，随机选取100粒籽粒测定粒重，进行3次重复，结果如图4所示，突变体的千粒重增幅达5.12～7.08％。随机选取20粒成熟收获后的籽粒，使用游标卡尺对籽粒粒宽进行测定，3次重复。结果如图5所示，两组OsBEE1突变体籽粒粒宽明显增加。
以上所述之实施例，只是本发明的较佳实施例而已，并非限制本发明的实施范围，故凡依本发明专利范围所述的构造、特征及原理所做的等效变化或修饰，均应包括于本发明申请专利范围内。
SEQUENCE LISTING
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tcccaagctc cacaacctgc caatgcagca gcagcagcca ttgtggagga tgcctcattg 180
gagagctcag tgtctgcagt tcttgacacc tctccatctg tggacaggaa gaggaaggca 240
gcagaagaca gtgcacactc caaggatagc tgcaaggatg gcaagagcag gagagggaag 300
aaggccagca aagaagtgga ggagaagagc accactgaag atgagcctcc aaaagggtac 360
atccatgtga gggcaaggag aggacaggca actgatagcc acagccttgc agagagggtg 420
aggagggaga ggatcagtga gaggatgagg atgctgcaag cactggtccc tggttgtgac 480
aaggttactg gaaaggctct cattttggat gagatcatca actatgtaca gtcgttgcag 540
aaccaagttg agttcctatc catgaggatt gcttcaatga gcccagtgtt gtatggcttt 600
ggaatggaca gcgatggcct ccatgaccaa aagattggag gcatgttcca agaagccctt 660
gcaatgccta atccagtact gaaccaatca agcccagctc catctcaagc tatcatggac 720
acaacctcaa ccacatccta ctcactgcaa agccagcatg gagccatctc tttctctcag 780
gacaatggca gttacctgat gcaagcagtg ggggagccaa ggcagcagga gatgctcaat 840
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gccaaacttg ctctactctt tagcctctgc caagaagaag tagtactcct cttttctctg 60
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ggagcagcca tcgccaccac caatgacccc aacatctcaa gcttcttcct ctacaaccac 240
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gcctcattgg agagctcagt gtctgcagtt cttgacacct ctccatctgt ggacaggaag 360
aggaaggcag cagaagacag tgcacactcc aaggtagcta acaactgaca caaccaaagc 420
attgacatta ttgacattgg cattcttgtt tgcatctctg aaagagaaca ttgttagttg 480
cattctggtt tctgaattct gggtttttgc aagcaggata gctgcaagga tggcaagagc 540
aggagaggga agaaggccag caaagaagtg gaggagaaga gcaccactga agatgagcct 600
ccaaaagggt acatccatgt gagggcaagg agaggacagg caactgatag ccacagcctt 660
gcagagaggg tactactact gcacttcact ttggttttgg ttttgatttt tctgctgctt 720
ttgcatgagt ggtttttggc ttgcagcttc tgcatctttt ctcctggtct ctgattcttg 780
gtgacatttt ctctctacag gtgaggaggg agaggatcag tgagaggatg aggatgctgc 840
aagcactggt ccctggttgt gacaaggtaa aaaataagtt ggttactatt taactattac 900
atgctctagt tcaattcttt cctctcattt ttatttttga gagtaattat tgttgagttg 960
tacagttgta gtgacatctt gctaatgcat ggctctaact ttgataagaa acaactgtgc 1020
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gttgcagaac caagttgagg tacactaaaa aaaaataaaa aaaagaagta atacttcatt 1140
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ccatgaccaa gtaaatatct aaataaatat ttgtaccttg aactaaatat gcctaaaatt 1320
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aagctatcat ggacacaacc tcaaccacat cctactcact gcaaagccag catggagcca 1500
tctctttctc tcaggttggc acatgtccac ctttctttgg ctaaatcata catgaaaatg 1560
tcccaattta gctacatata tcatggcatc aaagtaaaaa aaatccaaca attttaccca 1620
cctgaggtca tcactgaatg tgtgcatccc ctttcaggac aatggcagtt acctgatgca 1680
agcagtgggg gagccaaggc agcaggagat gctcaatcaa ttggtgttca acaacatgtg 1740
ctctttccag taggggaaga gtgacagaga aagcaagaaa aaaggacccc acattgacaa 1800
taggtactgc aaccaaagtt cattaattgt gactttgtga gaccctgaat attggattta 1860
ctactgtaaa ttttctctta actgcttctt ctaatatata aaagtcttct gaaattttga 1920
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caagctccac aacctgccaa tgcagcagca gcagccattg tggaggatgc ctcattggag 180
agctcagtgt ctgcagttct tgacacctct ccatctgtgg acaggaagag gaaggcagca 240
gaagacagtg cacactccaa ggatagctgc aaggatggca agagcaggag agggaagaag 300
gccagcaaag aagtggagga gaagagcacc actgaagatg agcctccaaa agggtacatc 360
catgtgaggg caaggagagg acaggcaact gatagccaca gccttgcaga gagggtgagg 420
agggagagga tcagtgagag gatgaggatg ctgcaagcac tggtccctgg ttgtgacaag 480
gttactggaa aggctctcat tttggatgag atcatcaact atgtacagtc gttgcagaac 540
caagttgagt tcctatccat gaggattgct tcaatgagcc cagtgttgta tggctttgga 600
atggacagcg atggcctcca tgaccaaaag attggaggca tgttccaaga agcccttgca 660
atgcctaatc cagtactgaa ccaatcaagc ccagctccat ctcaagctat catggacaca 720
acctcaacca catcctactc actgcaaagc cagcatggag ccatctcttt ctctcaggac 780
aatggcagtt acctgatgca agcagtgggg gagccaaggc agcaggagat gctcaatcaa 840
ttggtgttca acaacatgtg ctctttccag tag 873
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atggcagact tctcaccaca ccactctctc ctcaaggcta ctgctgctgg agcagccatc 60
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caagctccac aacctgccaa tgcagcagca gcagccattg tggaggatgc ctcattggag 180
agctcagtgt ctgcagttct tgacacctct ccatctgtgg acaggaagag gaaggcagca 240
gaagacagtg cacactccaa ggatagctgc aaggatggca agagcaggag agggaagaag 300
gccagcaaag aagtggagga gaagagcacc actgaagatg agcctccaaa agggtacatc 360
catgtgaggg caaggagagg acaggcaact gatagccaca gccttgcaga gagggtgagg 420
agggagagga tcagtgagag gatgaggatg ctgcaagcac tggtccctgg ttgtgacaag 480
gttactggaa aggctctcat tttggatgag atcatcaact atgtacagtc gttgcagaac 540
caagttgagt tcctatccat gaggattgct tcaatgagcc cagtgttgta tggctttgga 600
atggacagcg atggcctcca tgaccaaaag attggaggca tgttccaaga agcccttgca 660
atgcctaatc cagtactgaa ccaatcaagc ccagctccat ctcaagctat catggacaca 720
acctcaacca catcctactc actgcaaagc cagcatggag ccatctcttt ctctcaggac 780
aatggcagtt acctgatgca agcagtgggg gagccaaggc agcaggagat gctcaatcaa 840
ttggtgttca acaacatgtg ctctttccag tag 873
<210> 9
<211> 290
<212> PRT
<213> 人工序列
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Met Ala Asp Phe Ser Pro His His Ser Leu Leu Lys Ala Thr Ala Ala
1 5 10 15
Gly Ala Ala Ile Ala Thr Thr Asn Asp Pro Asn Ile Ser Ser Phe Phe
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35 40 45
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50 55 60
Ala Val Leu Asp Thr Ser Pro Ser Val Asp Arg Lys Arg Lys Ala Ala
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Glu Asp Ser Ala His Ser Lys Asp Ser Cys Lys Asp Gly Lys Ser Arg
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Arg Gly Lys Lys Ala Ser Lys Glu Val Glu Glu Lys Ser Thr Thr Glu
100 105 110
Asp Glu Pro Pro Lys Gly Tyr Ile His Val Arg Ala Arg Arg Gly Gln
115 120 125
Ala Thr Asp Ser His Ser Leu Ala Glu Arg Val Arg Arg Glu Arg Ile
130 135 140
Ser Glu Arg Met Arg Met Leu Gln Ala Leu Val Pro Gly Cys Asp Lys
145 150 155 160
Val Thr Gly Lys Ala Leu Ile Leu Asp Glu Ile Ile Asn Tyr Val Gln
165 170 175
Ser Leu Gln Asn Gln Val Glu Phe Leu Ser Met Arg Ile Ala Ser Met
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Ser Pro Val Leu Tyr Gly Phe Gly Met Asp Ser Asp Gly Leu His Asp
195 200 205
Gln Lys Ile Gly Gly Met Phe Gln Glu Ala Leu Ala Met Pro Asn Pro
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Val Leu Asn Gln Ser Ser Pro Ala Pro Ser Gln Ala Ile Met Asp Thr
225 230 235 240
Thr Ser Thr Thr Ser Tyr Ser Leu Gln Ser Gln His Gly Ala Ile Ser
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Phe Ser Gln Asp Asn Gly Ser Tyr Leu Met Gln Ala Val Gly Glu Pro
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Arg Gln Gln Glu Met Leu Asn Gln Leu Val Phe Asn Asn Met Cys Ser
275 280 285
Phe Gln
290

Claims (9)

1.OsBEE1基因在提高水稻产量中的应用，其特征在于，敲除水稻的OsBEE1基因增加粒宽，提高水稻的粒重；所述OsBEE1基因的核苷酸编码序列如SEQ ID NO:1所示。
2.敲除OsBEE1基因的株系在提高水稻产量中的应用，其特征在于，使用敲除OsBEE1基因的株系增加水稻的粒宽，提高水稻的粒重；所述OsBEE1基因的核苷酸编码序列如SEQ IDNO:1所示。
3.根据权利要求2所述的应用，其特征在于，所述敲除OsBEE1基因的株系为OsBEE1基因缺失纯合体。
4.敲除OsBEE1基因的试剂盒在提高水稻产量中的应用，其特征在于，敲除OsBEE1基因的试剂盒中包含以OsBEE1基因为靶点的CRISPR/Cas9-gRNA表达载体；所述OsBEE1基因的核苷酸编码序列如SEQ ID NO:1所示。
5.根据权利要求4所述的应用，其特征在于，使用敲除OsBEE1基因的试剂盒增加水稻的粒宽、提高水稻的粒重。
6.根据权利要求4所述的应用，其特征在于，所述gRNA的靶点序列为5′-ACACCACTCTCTCCTCCTCAAGG- 3′，PAM序列为3′端AGG。
7.根据权利要求2或6所述的应用，其特征在于，敲除OsBEE1基因的方法，包括如下步骤：
（1）gRNA靶点序列的选择：
选择的靶点序列为5′- ACACCACTCTCTCCTCCTCAAGG- 3′，所述PAM序列为3′端AGG；
（2）gRNA寡核苷酸链上下游引物的设计：
上游引物为BEE1_gRNA_F: 5′- GGCGACACCACTCTCTCCTCCTCA-3′，
下游引物为BEE1_gRNA_R: 5′- AAACTGAGGAGGAGAGAGTGGTGT-3′；
（3）gRNA表达载体构建：
将gRNA寡核苷酸链上下游引物混合、退火，得寡核苷酸双链DNA；用内切酶BsaI酶切CRISPR/Cas9质粒，得线性质粒；用T4连接酶将所述线性质粒和寡核苷酸双链DNA连接，得连接产物；将连接产物转化、筛选、验证，即得CRISPR/Cas9-gRNA表达载体；
（4）将CRISPR/Cas9-gRNA表达载体导入农杆菌中，得CRISPR/Cas9-gRNA农杆菌；用CRISPR/Cas9-gRNA农杆菌侵染水稻愈伤组织；
（5）将步骤（4）获得的愈伤组织诱导得到再生苗，筛选得到转基因阳性植株，鉴定即得水稻OsBEE1突变体。
8.根据权利要求7所述的应用，其特征在于，选择所述水稻OsBEE1突变体中的纯合突变体用于提高水稻产量。
9.根据权利要求8所述的应用，其特征在于，选择T0代双等位突变体自交得T1代，从包括T1代的后继世代中选择纯合突变体用于提高水稻产量。
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