WO2023202038A1 - 调控玉米根系夹角和倒伏抗性的基因及其应用 - Google Patents
调控玉米根系夹角和倒伏抗性的基因及其应用 Download PDFInfo
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Definitions
- This application belongs to the field of biological gene technology, and specifically relates to a gene that regulates corn root angle and lodging resistance and its application.
- Corn is an important crop that integrates food, feed, and industrial raw materials. It is also the crop with the highest productivity in the world. Its sufficient and stable supply is crucial to ensuring food security around the world.
- the situation of corn lodging hazards has become increasingly severe. Lodging has become the main limiting factor for high and stable corn yields. .
- Corn lodging is a phenomenon in which corn roots or stems bend or break due to external force.
- the main hazards are: 1) Lodging disrupts the spatial order of leaves, causing plant collisions and damaging leaf tissue, resulting in weakened photosynthetic efficiency of plants and affecting yields. . 2) Lodging destroys the rhizome conduction system, affecting the transportation of nutrients, water and photosynthetic products, resulting in reduced yields. 3) Lodging will cause ear sprouting, aggravate the occurrence of ear diseases, and affect the quality of corn. 4) Lodging will cause confusion in plant arrangement, greatly increasing the difficulty and cost of harvesting.
- Root lodging is usually divided into two types: root lodging and stem lodging. Root lodging occurs almost throughout the entire corn growth period, and its occurrence range is wider. It is the most important lodging disaster affecting corn production. Research shows that the structure of the root system is the main reason affecting corn root collapse.
- the root system is the most important organ for fixing plant plants and obtaining underground nutrients; the root system of corn is mainly composed of two parts: the radicle root system and the nodal root system.
- the radicle system is mainly composed of primary roots and seed roots. It reaches its maximum in the V2 stage (the second fully expanded leaf stage) and is the main organ for fixing the corn plant in the seedling stage and obtaining water and underground nutrients.
- the node root system mainly refers to the roots growing on the corn stem nodes, which mainly includes the crown roots growing on the underground nodes and the aerial roots growing on the above-ground nodes.
- Aerial roots can "grasp the ground” to form a cone-shaped structure, effectively supporting the corn plant to stand upright; and generally, the top two layers of aerial roots of corn (generally born on the 6th-7th node of the stem) can occupy the node 50% of the total root mass is the most important functional root system of corn. Therefore, aerial roots are the most important organ that affects corn root lodging resistance and nutrient absorption capacity.
- the purpose of the embodiments of this application is to provide a gene ZmYUC2 and ZmYUC4 that regulates the angle of corn aerial roots and lodging resistance and its application, as well as a method for enhancing the lodging resistance of corn.
- the embodiments of the present application provide a gene that regulates the angle of the corn root system and lodging resistance. After mutation of the gene in the plant, it has the ability to increase the growth angle of the aerial roots of corn and enhance the lodging resistance of the corn plant.
- the multi-nucleation of the gene The nucleotide sequence is selected from one of the following groups:
- the increasing angle between aerial roots or the increasing angle of aerial root growth described in the embodiments of this application is the same concept, which refers to the increasing angle between the aerial roots and the corn stalk. This increased angle forms a "cone shape”. ”, which enhances the lodging resistance of corn plants.
- the percentage of sequence similarity described in this application can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithms, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm, which are well known to those skilled in the art.
- the embodiments of the present application provide an expression cassette, recombinant vector or cell, which contains part or all of a gene sequence for regulating corn root angle and lodging resistance.
- the sequence of the gene One of the polynucleotide sequences selected from the following group:
- the embodiments of the present application also provide a gene mutant sequence, which is obtained by mutation of the genome nucleotide sequence or promoter sequence of the gene.
- Corn plants containing the gene mutant sequence have the characteristics of enlarged aerial root angle and resistance to lodging.
- Phenotype the nucleotide sequence of the gene is selected from one of the following groups of sequences:
- the sequence is a fragment of the nucleotide sequence shown in SEQ ID No: 1, 3, 5, 9 or 10 that conforms to the 5'-Nx-NGG-3' sequence arrangement rules, where N represents A, G, C and Any of T, 14 ⁇ X ⁇ 30, and X is an integer, Nx represents X consecutive nucleotides; or
- the embodiments of the present application also provide mutants of lodging resistance genes ZmYUC2 and ZmYUC4.
- the two mutation types of the ZmYUC2 gene are the deletion of 1288bp-1292bp (ATTGC) downstream of the start codon (ATG) on the genomic DNA sequence and the insertion of an A base between 1285bp-1286bp;
- two types of ZmYUC4 gene are the 254bp or 255bp base (A) deletion and the downstream 937bp or 938bp base (G) deletion downstream of the start codon (ATG) on the genomic DNA sequence, and the 253bp-267bp (GAAGACTACCCGGAG) deletion and the downstream 936bp -937bp(CG) deletion.
- the reduction or inhibition of the normal expression or protein function of lodging-related genes includes obtaining it through mutation.
- the mutation includes the substitution, deletion and/or addition of one or more nucleotides on the nucleotide sequence or promoter sequence of the gene, so that the aerial root angle of the plant containing the mutation becomes larger, Enhance the ability to resist lodging.
- the mutation includes, but is not limited to, obtained by physical mutagenesis, chemical mutagenesis, gene editing and other methods.
- Physical mutagenesis includes but is not limited to radiation mutagenesis, space breeding, etc.;
- chemical mutagenesis methods include mutagenesis caused by treatment with mutagens such as EMS;
- gene editing methods include but is not limited to ZFN, TALEN and/or CRISPR/ Cas and other methods.
- nucleotide sequence after mutation of the gene is as shown in any one of SEQ ID Nos: 11-14.
- Stringent hybridization conditions means conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence to a more detectable extent (e.g., at least 2-fold above background) than to other sequences. Stringent hybridization conditions are sequence-dependent and vary in different environments. Conditions will vary, with longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. More specifically, the stringent conditions are usually Choose to be about 5-10°C below the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm is the temperature at which a probe complementary to the target is 50% hybridized to the target sequence at equilibrium.
- Tm thermal melting point
- seed root several roots that grow from the original embryo.
- node root refers to the roots growing on the nodes of corn stems, including crown roots and aerial roots (brace roots).
- Figure 3 shows in situ hybridization analysis; ZmYUC2 is mainly expressed in the resting center of the root tip and near the root cap, and ZmYUC4 is mainly expressed in the root cap part of the root tip; the tissue used is the root tip of the aerial root of maize.
- FIG. 4 shows CRISPR/Cas9 gene editing and mutant analysis.
- A shows the design of ZmYUC2 and ZmYUC4 gene editing target sites;
- B shows the genotype identification of single mutants and double mutants of Zmyuc2 and Zmyuc4.
- Figure 6 shows the comparative analysis of the amino acid sequences of different mutants of Zmyuc4 and the wild-type amino acid sequences.
- Figure 7 shows the phenotypic analysis of the aerial root angle of the Zmyuc2 and Zmyuc4 gene-edited mutants;
- A is a picture of the field root phenotypes of the wild type (WT) and mutants during the silking stage, and the white ruler is 15cm.
- B-E shows the number of aboveground aerial roots (BR) of the wild type and various Zmyuc mutants grown in the field during the silking stage (B), the number of top BR roots (C), and the BR diameter (D) and analysis of BR growth angle (E); n>20.
- F X-ray CT images of wild type and various Zmyuc mutants grown in soil to the V6 stage. The red arrow indicates above-ground stem rooting, and the yellow scale is 5cm.
- G-H Stem root number (G) and angle (H) measured from X-ray CT images;
- Figure 11 shows the effect of ZmYUC2 and ZmYUC4 gene mutations on the local auxin content of the root tip and its response to gravity;
- a and C In the control material CK, and different mutant materials of Zmyuc2 and Zmyuc4, in the root tips growing along the direction of gravity, Comparison of RFP fluorescence intensity (A) and its statistics (C);
- B Comparison of RFP fluorescence intensity in root tips growing in the vertical direction of gravity in the control material CK, and different mutant materials of Zmyuc2 and Zmyuc4; D.
- the inventors used the reported YUC3, YUC5, YUC7, YUC8 and YUC9 protein sequences in Arabidopsis (Chen et al.
- the ZmYUC4 gene (its genomic nucleotide sequence is shown as SEQ ID No:10) has only one transcript
- the ZmYUC2 gene (its genomic DNA sequence is shown as SEQ ID No:9) has two transcripts ZmYUC2 -T001 (the nucleotide sequence of which is shown in SEQ ID No: 1) and ZmYUC2-T002 (the nucleotide sequence of which is shown in SEQ ID No: 3).
- Example 2 In situ hybridization experiments show that ZmYUC2 is mainly expressed in the resting center and root cap of corn root tips, and ZmYUC4 is mainly expressed in the root cap of corn root tips.
- the aerial root angle and ground root coverage of the Zmyuc2/Zmyuc4 double mutant are significantly larger than those of the wild type:
- the two mutation types of the ZmYUC2 gene are the deletion of 1288bp-1292bp (ATTGC) downstream of the start codon (ATG) on the genomic DNA sequence and the insertion of an A base between 1285bp-1286bp; the ZmYUC4 gene
- the two mutation types are the deletion of the 255bp base (A) downstream of the start codon (ATG) and the deletion of the downstream 938bp base (G) of the genomic DNA sequence, and the deletion of the 253bp-267bp (GAAGACTACCCGGAG) and the downstream 936bp-937bp. (CG) is missing.
- Amino acid sequence analysis showed that the deletion and insertion of the aforementioned bases resulted in premature termination or frameshift mutation of the amino acid sequence coding of the ZmYUC2 and/or ZmYUC4 genes in the mutants, in which both transcripts of the ZmYUC2 gene were mutated.
- the amino acid sequence of the protein encoded by the mutation and its comparison with the wild type are shown in Figure 5.
- the amino acid sequence of the Zmyuc4 mutant and its comparison with the wild type are shown in Figure 6.
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
提供了调控玉米根系夹角和倒伏抗性的ZmYUC2和ZmYUC4基因及其应用,该ZmYUC2和ZmYUC4基因能够特异的调控根尖局部的生长素合成,调控玉米根系的夹角的同时对其余农艺性状无不利影响,进而可应用于抗倒伏育品种。
Description
本申请要求于2022年4月22日提交中国专利局、优先权号为202210423670.9、发明名称为“调控玉米根系夹角和倒伏抗性的基因及其应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本申请属于生物基因技术领域,具体涉及一种调控玉米根系夹角和倒伏抗性的基因及其应用。
玉米是集粮食、饲料、工业原料于一身的重要农作物,也是世界上生产能力最高的农作物,其充足稳定的供应对保证世界范围内的粮食安全至关重要。近年来由于气候环境的恶化、各种逆境胁迫和灾害的加剧、氮肥的大规模施用、密植栽培的推行等原因,玉米倒伏危害的形势日益严峻,倒伏已经成为当前玉米高产、稳产的主要限制因子。
玉米倒伏是由于外力引发的玉米根或茎秆弯倒或折断的现象,其危害主要表现在:1)倒伏打乱叶片的空间秩序,造成植株碰撞破坏叶片组织,导致植株光合效率减弱,影响产量。2)倒伏破坏根茎输导系统,影响养分、水分及光合产物的运输,造成减产。3)倒伏会造成穗发芽,加重穗部病害发生,影响玉米品质。4)倒伏会造成植株排列的错乱,大大增加收获难度和成本。已有的统计数据显示,玉米倒伏可造成减产达15-50%,严重时甚至造成玉米绝收;玉米倒伏率每增加1%,大约减产108kg/hm。调查也显示,包括产量在内的所有性状中,抗倒是农民最关注的性状,是农民选取品种的首要参考因素。因此,良好的倒伏抗性是玉米新品种选育的首要育种目标。
玉米倒伏通常分为2种类型:根倒、茎倒。根倒的发生几乎贯穿了整个玉米生长期,其发生的范围更加广泛,是影响玉米生产的最主要倒伏灾害。研究表明,根系的构型是影响玉米根倒的主要原因。根系是植物植株固定及获取地下营养的最主要的器官;玉米的根系主要由胚根系和节根系两部分组成。胚根系主要由初生根和种子根组成,其在V2时期(第二片完全展开叶时期)达到最大,是幼苗期玉米植株固定、获取水分和地下营养的主要器官。节根系主要指玉米茎节上着生的根,其主要包括地下节上着生的冠根、及地上节上着生的气生根。气生根可以“抓地”形成锥形结构,有效的支撑玉米植株直立;并且一般情况下,玉米最上边两层气生根(一般着生于第茎秆的第6-7节)可占到节根总量的50%,是玉米的最主要功能根系。因此,气生根是影响玉米根倒抗性及养分吸收能力的最主要器官。
因为玉米根系系统复杂,相关表型测量困难、成本高,易受环境影响等原因,当前我国乃至世界上玉米节根发育及构型调控的遗传基础研究、以及抗倒伏基因的克隆和功能研究还相对滞后。
发明内容
本文提到的所有参考文献都通过引用并入本文。除非有相反指明,本文所用的所有技术和科学术语都具有与本发明所属领域普通技术人员通常所理解的相同的含义。除 非有相反指明,本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用,而非加以限制。
本申请实施例的目的在于提供一种调控玉米气生根夹角和倒伏抗性相关的基因ZmYUC2和ZmYUC4及其应用,以及增强玉米抗倒伏性的方法。
本申请实施例提供了一种调控玉米根系夹角和倒伏抗性的基因,植株中的该基因突变后具有使玉米气生根生长角度变大、增强玉米植株抗倒伏的能力,所述基因的多核苷酸序列选自下列组的序列之一:
(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;
(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;
(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有增大玉米气生根生长角度和增强倒伏抗性的功能;
(d)与(a)-(c)任一所示的多核苷酸序列至少有90%、95%、98%或以上相似性的多核苷酸序列,该多核苷酸序列的突变具有使玉米气生根生长角度变大和抗倒伏的功能;或
(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
本申请实施例中所述的气生根夹角变大或气生根生长角度变大是同一概念,指的是气生根与玉米茎秆之间的角度变大,该增大的角度形成“锥形”,增强了玉米植株的抗倒伏能力。
本申请实施例所提供的基因,还包括与本发明实施例所公开的倒伏相关基因的核苷酸序列有至少80%、85%、90%、95%、98%或99%序列相似性的同源基因,或者与本发明实施例所公开的倒伏相关基因的氨基酸序列有至少90%、95%或98%序列相似性的同源基因,且玉米中所述同源基因突变后具有使其气生根生长角度变大,从而增强植株的抗倒伏能力的功能,可以从任何玉米品种中分离获得。
本领域技术人员应该知晓,同一种植物不同品种间的同一基因存在单核苷酸多样性(single nucleotide polymorphism,SNP),即同一基因的核苷酸序列往往存在个别碱基的差异,但同一作物品种数量很多,发明人不可能进行一一列举,本申请实施例仅提供了玉米作物中具有代表性的品种的序列。因此,本领域技术人员应该知悉,不同品种来源的与本发明所保护基因及其核苷酸序列存在SNP的核苷酸序列也在本发明的保护范围内。
本申请中所述的序列相似性的百分比可以通过公知的生物信息学算法来获得,包括Myers和Miller算法、Needleman-Wunsch全局比对法、Smith-Waterman局部比对法、Pearson和Lipman相似性搜索法、Karlin和Altschul的算法,这对于本领域技术人员来说是公知的。
本申请实施例提供了一种表达盒、重组载体或细胞,所述表达盒、重组载体或细胞含有用于调控玉米根系夹角和倒伏抗性的基因序列的部分或全部,所述基因的序列选自下列组的多核苷酸序列之一:
(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;
(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;
(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有调控玉米根系夹角和倒伏抗性的功能;
(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列;
(e)具有(a)-(d)任一所示多核苷酸序列中300或500个以上连续多核苷酸序列的序列;或
与(a)-(e)之任一所述序列互补的多核苷酸序列。
本申请实施例还提供了一种基因突变体序列,由基因的基因组核苷酸序列突变或是启动子序列突变获得,含有该基因突变体序列的玉米植株具有气生根夹角变大和抗倒伏的表型,所述基因的核苷酸序列选自下列组的序列之一:
(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;
(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;
(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有调控玉米根系夹角和倒伏抗性的功能;
(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列;或
(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
可选地,所述基因启动子的突变体序列,是指通过突变启动子的核苷酸序列获得的突变体序列,该突变可使相应启动子的转录功能降低,从而降低该启动子所驱使基因的表达。优选地,所述突变发生在启动子的保守序列区。
可选地,所述基因突变体序列或启动子突变体序列通过突变的方式获得,所述突变包括在该基因或启动子的核苷酸序列上进行一个或多个核苷酸的取代、缺失和/或添加。
具体地,所述突变可以通过物理诱变、化学诱变或基因编辑的方式获得。物理诱变包括但不限于辐射诱变、太空育种等;化学诱变的方法包括用EMS等诱变剂处理所导致的诱变;基因编辑的方法包括但不限于ZFN、TALEN和/或CRISPR/Cas等方法。
本领域技术人员知悉,CRISPR/Cas基因编辑系统或基因编辑方法的主要原理是通过一个叫向导RNA(guide-RNA,gRNA)的核酸片段在宿主基因组找到要进行基因编辑的位置,也就是靶向DNA序列,然后通过Cas蛋白对DNA进行切割。在本申请中,所述Cas蛋白包括但不限于Cas9、Cas12、Cas12a、Cas12j、Cas12e、Cas13和/或Cas14等蛋白。
可选地,在所使用的基因编辑系统为CRISPR/Cas9时,所述通过CRISPR/Cas9方法获得的基因突变体序列,其CRISPR/Cas9技术所用的靶点序列选自下列组的序列之一:
(a)序列为SEQ ID No:1、3、5、9或10所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段,其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸;或
(b)与(a)所述的多核苷酸序列互补的核苷酸序列。
本申请实施例还提供了抗倒伏基因ZmYUC2和ZmYUC4的突变体。具体地,ZmYUC2基因的两种突变类型分别为基因组DNA序列上起始密码子(ATG)下游的第1288bp-1292bp(ATTGC)缺失和第1285bp-1286bp间插入一个A碱基;ZmYUC4基因的两种突变类型分别为基因组DNA序列上起始密码子(ATG)下游的第254bp或255bp碱基(A)缺失及下游937bp或938bp碱基(G)缺失,第253bp-267bp(GAAGACTACCCGGAG)缺失及下游936bp-937bp(CG)缺失。
可选地,本发明实施例所提供的基因突变体,其核苷酸序列如SEQ ID No:11-14之任一所示。
本申请实施例还提供了一种增强玉米抗倒伏能力的方法,所述方法通过降低或抑制倒伏相关基因的正常表达或蛋白功能,使玉米植株气生根夹角变大,增强抗倒伏能力,所述基因的多核苷酸序列选自下列组的序列之一:
(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;
(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;
(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,抑制该多核苷酸序列的表达具有使玉米气生根生长角度变大和抗倒伏的功能;
(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列,抑制该多核苷酸序列的表达具有使玉米气生根生长角度变大和抗倒伏的功能;或
(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
可选地,本申请实施例所述的增强玉米抗倒伏能力的方法,其中所述的降低或抑制倒伏相关基因的正常表达或蛋白功能,包括通过RNA干扰(即RNAi)和/或突变的方式获得。本领域技术人员知晓,RNAi技术为本领域的常规技术,其通过21-23bp的短链双链RNA(siRNA∶smallinterfering RNA)或者是长链双链RNA(dsRNA∶double-strand RNA)与目的基因表达的mRNA同源区进行特异性结合,使mRNA降解,达到抑制基因表达的作用。
可选地,在本申请中可以通过RNAi的方法抑制本申请ZmYUC2和/或ZmYUC4倒伏相关基因的表达,从而影响基因的活性,使玉米植株获得气生根夹角增大和抗倒伏的表型。
可选地,本申请实施例所述的增强玉米抗倒伏能力的方法,其中所述的降低或抑制倒伏相关基因的正常表达或蛋白功能,包括通过突变的方式获得。所述突变包括在该基因的核苷酸序列上或启动子序列上进行一个或多个核苷酸的取代、缺失和/或添加,从而使含有该突变的植株具有气生根夹角变大,增强抗倒伏能力的功能。
可选地,所述突变包括但不限于通过物理诱变、化学诱变、基因编辑等方法获得。物理诱变包括但不限于辐射诱变、太空育种等;化学诱变的方法包括用EMS等诱变剂处理所导致的诱变;基因编辑的方法包括但不限于ZFN、TALEN和/或CRISPR/Cas等方法。
可选地,本申请实施例所述的增强玉米抗倒伏能力的方法,在所使用的基因编辑系统为CRISPR/Cas9时,在使用CRISPR/Cas9进行基因或基因的启动子编辑时,所述CRISPR/Cas9方法所用的靶点序列选自下列组的序列之一:
(a)序列为SEQ ID No:1、3、5、7、8、9或10所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段,其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸;或
(b)与(a)所述的多核苷酸序列互补的核苷酸序列。
可选地,所述基因突变后的核苷酸序列如SEQ ID No:11-14之任一所示。
本申请实施例还提供了一种非作为繁殖材料的植物细胞、组织、器官或产品,所述植物细胞、组织、器官或产品含有本申请实施例任一所述的突变体序列。
本申请实施例还提供了本发明任一实施例所公开的基因、表达盒、重组载体或细胞、方法、及其获得的突变体材料或转化事件在育种中的应用。
可选地,所述在育种中的应用是指通过RNAi、基因突变、启动子突变和/或与突变体材料杂交的方式,使玉米植株抗倒伏能力增强的方法。
可选地,本发明实施例所述的在根尖局部的静止中心和根冠组织中表达的启动子在调控基因特异地在根尖的静止中心和根冠部位表达方面的应用。
可选地,本发明实施例所述的在根尖局部的根冠组织中表达的启动子在调控基因特异地在根尖的根冠部位表达方面的应用。
可选地,本发明实施例所述的一种改良玉米根系角度和倒伏抗性的方法,将ZmYUC2和ZmYUC4基因进行突变,或表达量敲低,或组织表达特异性改变。
本申请实施例的有益效果是:本申请实施例所提供的倒伏相关基因ZmYUC2和/或ZmYUC4能够调控玉米根尖局部的生长素合成,来调控玉米根系向重力性,进而调控玉米气生根夹角,并增强玉米植株的抗倒伏性,且对其他农艺性状无不良影响。本申请实施例所提供的倒伏相关基因、突变体及其应用方法,对玉米抗倒伏育种具有重要意义。
本申请所涉及到的部分术语定义:
本申请中所述“严谨杂交条件”意指在所属领域中已知的低离子强度和高温的条件。通常,在严谨条件下,探针与其靶序列杂交的可检测程度比与其它序列杂交的可检测程度更高(例如超过本底至少2倍。严谨杂交条件是序列依赖性的,在不同的环境条件下将会不同,较长的序列在较高温度下特异性杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100%互补的靶序列。更具体的,所述严谨条件通常被选择为低于特异序列在规定离子强度pH下的热熔点(Tm)约5-10℃。Tm为在平衡状态下50%与目标互补的探针杂交到目标序列时所处的温度。严谨条件可为以下条件:其中在pH7.0到8.3下盐浓度低于约1.0M钠离子浓度,通常为约0.01到1.0M钠离子浓度,并且温度对于短探针(包括但不限于10到50个核苷酸)而言为至少约30℃,而对于长探针(包括但不限于大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言,正信号可为至少两倍的背景杂交,视情况为10倍背景杂交。例示性严谨杂交条件可如下:50%甲酰胺,5×SSC和1%SDS,在42℃下培养;或5×SSC,1%SDS,在65℃下培养,在0.2×SSC中洗涤和在65℃下于0.1%SDS中洗涤。所述洗涤可进行5、15、30、60、120分钟或更长时间。
术语“胚根”:玉米种子萌发后由胚生组织直接产生的根,包括初生根(primary root)和种子根(seminal roots)。
术语“初生根”:最初从萌发的种子中生长出来的那一条根。
术语“种子根”:从最初的胚中生长出来的几条根。
术语“节根”:玉米茎节上着生的根,其包括冠根(crown roots)和气生根(brace roots)。
术语“冠根”:玉米地下节上着生的节根。术语“气生根”:又叫“支撑根”,玉米地上节上着生的节根。术语“基因”在本申请中被定义为包含一个或多个多核苷酸的遗传单位,该遗传单位占据染色体或质粒上特定位置并且含有用于生物中的特定特征或性状的遗传指令。
术语“RNA干扰”,(RNAinterference,RNAi),是一门基因阻断技术,为一种双链RNA(double-strandedRNA,dsRNA)分子在mRNA水平上阻断特异基因的表达或使其沉默的过程,即序列特异性的转录后基因沉默(Post-transcriptional gene silencing, PTGS)。
图1为玉米和拟南芥YUC蛋白的系统进化树;箭头所指的即为ZmYUC2和ZmYUC4基因所编码蛋白。
图2为ZmYUC2和ZmYUC4基因的RT-qPCR分析;结果表明ZmYUC2和ZmYUC4主要在玉米根系组织中表达。
图3为原位杂交分析;ZmYUC2主要在根尖的静止中心及根冠附近表达,ZmYUC4主要在根尖的根冠部位表达;所用组织为玉米气生根的根尖。
图4为CRISPR/Cas9基因编辑及突变体分析。其中A为ZmYUC2和ZmYUC4基因编辑靶位点设计情况;B为Zmyuc2和Zmyuc4的单突变体以及双突变体基因型鉴定。
图5为Zmyuc2不同突变体氨基酸序列与野生型氨基酸序列的比对分析。
图6为Zmyuc4不同突变体氨基酸序列与野生型氨基酸序列的比对分析。
图7为Zmyuc2和Zmyuc4基因编辑突变体的气生根夹角表型分析;其中(A)为野生型(WT)和突变体在吐丝期的田间根系表型图片,白色标尺为15cm。(B-E)为吐丝期时对在田间生长的野生型和各种Zmyuc突变体地上部气生根(brace roots,BR)轮数(B)、顶部BR根数(C)、BR直径(D)和BR生长角(E)的分析;n>20。(F)为在土壤中生长到V6期的野生型和各种Zmyuc突变体的X-ray CT图像。红色箭头表示地上的茎生根,黄色标尺为5cm。(G-H)从X-ray CT图像测量的茎根数(G)和角度(H);
n>10。
图8为田间密度种植试验分析表明高密度种植条件下Zmyuc2/Zmyuc4双突变体的根倒抗性较野生型对照材料显著增强;A.2021年廊坊Zmyuc2/Zmyuc4双突变体及野生型对照材料的抽雄期根倒推力对比;横坐标为茎秆与偏离垂直线的角度,纵坐标为将茎秆推到一定角度所用的力;B.高密度下根系倒伏的图像(135000株/公顷)。上图是搭载1/1.3英寸(4800万像素)图像传感器相机的无人直升机拍摄的航拍照片。C.WT与Zmyuc2和Zmyuc4单基因突变体及双基因突变体在高密度下的根系倒伏分析;Leve l1:倒伏程度≤30°。Level 2为30°<倒伏度≤60°。Level 3表示倒伏程度>60°。
图9为田间表型观察表明Zmyuc2和Zmyuc4的单突变和双突变不会造成植株高度、叶片构型等植株构型相关性状的明显改变。
图10为田间表型观察表明Zmyuc2和Zmyuc4的单突变和双突变不会造成穗部大小、籽粒及产量相关性状的明显改变。
图11为ZmYUC2和ZmYUC4基因突变后影响根尖局部的生长素含量及对重力的响应;A和C.对照材料CK、及Zmyuc2和Zmyuc4不同突变体材料中,沿重力方向生长的根尖中,RFP荧光强度对比(A)及其统计情况(C);B.对照材料CK、及Zmyuc2和Zmyuc4不同突变体材料中,垂直重力方向生长的根尖中,RFP荧光强度对比情况;D.对照材料CK、及Zmyuc2和Zmyuc4不同突变体材料中,垂直重力方向生长的根尖的上下表皮(B图中箭头所指部位)中RFP荧光强度的统计情况;整体上,根据RFP荧光强度的变化可知,Zmyuc2和Zmyuc4突变体中根冠的生长素含量降低,并影响重力刺激后的根尖生长素含量分布。
图12为ZmYUC2基因在中国现代玉米育种过程中受到了显著的人工选择;A.中国黄早四亚群不同年代材料间的气生根表型的比较分析,**表示有极显著差异;ns表示无显著差异;B.ZmYUC2基因区域选择信号(XP-CLR方法)概况;图中“<”表示ZmYUC2 基因的位置;最上边的水平虚线表示的是全基因组最高2%选择信号的显著性阈值;图上半部分别表示中国不同年代自交系间的选择信号情况,下半部为黄早四亚群不同年代材料间的选择信号情况。
为了便于理解本申请,下面将对本申请进行更全面的描述。但是,本申请实施例可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本申请实施例的公开内容的理解更加透彻全面。
以下实施例中所用的自交系可从“中国作物种质信息网”得到相关信息和申请获取对应的种子。
实施例1、基因表达分析表明ZmYUC2和ZmYUC4主要在玉米根系组织中特异表达1.同源蛋白分析表明玉米基因组中存在14个编码YUC蛋白的同源基因:
发明人以拟南芥中已报道的YUC3、YUC5、YUC7、YUC8和YUC9蛋白序列(Chen et
al.,2014)为查询目标(Query),在gramene数据库中的玉米序列数据库B73AGPv4(http://ensembl.gramene.org/Zea_mays/Info/Index)中进行比对(BLASTP)搜索(E-value选择1e-10,其它参数为默认值),共得到了14个YUC同源基因(图1),分别命名为:SPI1、DE18、ZmYUC2、ZmYUC3、ZmYUC4、ZmYUC5、ZmYUC6、ZmYUC7、ZmYUC8、ZmYUC9、ZmYUC10、ZmYUC11、ZmYUC12、和ZmYUC13(图1)。
进一步分析发现,ZmYUC4基因(其基因组核苷酸序列如SEQ ID No:10所示)只有一个转录本,而ZmYUC2基因(其基因组DNA序列如SEQ ID No:9所示)有两个转录本ZmYUC2-T001(其核苷酸序列如SEQ ID No:1所示)和ZmYUC2-T002(其核苷酸序列如SEQ ID No:3所示)。通过转录组数据分析表明ZmYUC2-T002,即SEQ ID No:3,是ZmYUC2基因的优势转录本,其长度比ZmYUC2-T001(SEQ ID No:1)多出102bp;而两个转录本仅在第1030bp之后存在差异,两个转录本所编码蛋白应该都能行使相应的功能。后续的分析都是基于第1030bp之前序列设计的引物及探针进行。
2.基因表达分析表明ZmYUC2和ZmYUC4主要在玉米根系组织中表达:
利用已发表的玉米全生育期基因表达数据(https://www.maizegdb.org/)绘制了ZmYUCs的基因表达热图。分析发现,有7个ZmYUC基因在玉米根系相关的组织中有表达,其中ZmYUC2和ZmYUC4具有较高的根系表达特异性(在根中优势表达,其他组织中表达较低或不表达,图2)。为了验证ZmYUC2和ZmYUC4的组织表达特异性,发明人对B73自交系V1时期的根、地上部分幼苗,V13时期的茎(第10节)、叶片(最上边展开叶)、幼嫩雌穗、幼嫩雄穗,授粉后15天的籽粒,进行了qRT-PCR分析,结果表明ZmYUC2和ZmYUC4确实主要在玉米根系组织中特异表达(图2),进一步预示ZmYUC2和ZmYUC4可能在调控玉米根系发育方面具有重要功能。
实施例2原位杂交实验表明ZmYUC2主要在玉米根尖的静止中心和根冠部位表达,ZmYUC4主要在根尖的根冠部位表达
为了确定ZmYUC2和ZmYUC4的具体组织表达部位,发明人分别设计了这两个基因的特异性探针,对B73自交系的幼嫩气生根根尖进行了原位杂交实验,结果表明(图3),ZmYUC2主要在玉米根尖的静止中心和根冠部位表达,ZmYUC4主要在根尖的根冠 部位表达。静止中心是控制周围的干细胞分化,维持根尖分生组织的活性的重要组织;根冠是植物根尖感受重力信号的关键组织。这些结果预示着ZmYUC2和ZmYUC4可能在玉米根系的向重力性调控方面具有重要作用。
实施例3、Zmyuc4单突变、Zmyuc2和Zmyuc4基因双突变均增大玉米的根系夹角、增加玉米的倒伏抗性,但不带来其余不利的影响
1.Zmyuc2/Zmyuc4双突变体的气生根角度及地面根系覆盖范围较野生型显著增大:
为了确定ZmYUC2和ZmYUC4的生物学功能,发明人构建了这两个基因的CRISPR/Cas9基因编辑载体(图4A),并遗传转化玉米自交系ZC01。通过对T1代转基因材料进行PCR检测及转基因载体分离,获得了不含CRISPR载体的Zmyucm2和Zmyuc4单突变体,Zmyuc2/Zmyuc4双突变体各两个株系(图4B),将其分别命名为:Zmyuc2#1、Zmyuc2#2、Zmyuc4#1、Zmyuc4#2、Zmyuc2/4#1、Zmyuc2/4#2。通过检测分析发现,ZmYUC2基因的两种突变类型分别为基因组DNA序列上起始密码子(ATG)下游的第1288bp-1292bp(ATTGC)缺失和第1285bp-1286bp间插入一个A碱基;ZmYUC4基因的两种突变类型分别为基因组DNA序列上起始密码子(ATG)下游的第255bp碱基(A)缺失及下游938bp碱基(G)缺失,第253bp-267bp(GAAGACTACCCGGAG)缺失及下游936bp-937bp(CG)缺失。氨基酸序列分析表明,前述碱基的缺失和插入,导致突变体中的ZmYUC2和/或ZmYUC4基因发生了氨基酸序列编码的提前终止或移码突变,其中ZmYUC2基因的两个转录本都发生了突变,其突变后编码的蛋白氨基酸序列及其与野生型的比对分析见图5,Zmyuc4突变体氨基酸序列及其与野生型的比对分析见图6。
2021年夏天,在河北廊坊试验站进行田间表型分析表明,Zmyuc2/Zmyuc4双突变体的气生根角度及地面根系覆盖范围较其野生型对照材料显著增大(图7A和7E)。2021年冬季在海南实验基地再次进行田间实验,确认了Zmyuc2/Zmyuc4双突变体气生根角度增大的表型;利用X-ray CT进一步比较单、双突变体的表型还发现,Zmyuc2单突变体的气生根夹角与野生型对照材料没有明显差异,Zmyuc4单突变体的气生根夹角比野生型对照材料的大,但显著小于双突变体的气生根夹角(图7F和7H);表明ZmYUC2和ZmYUC4在气生根夹角调控方面存在功能冗余。进一步调查分析发现,Zmyuc2和Zmyuc4基因编辑突变体的气生根数目、冠根数目与野生型对照材料没有显著差异(图7B-D和图7G),表明ZmYUC2和ZmYUC4可能具有气生根夹角的特异性的调控作用。
2.Zmyuc4单突变体及Zmyuc2/Zmyuc4双突变体的倒伏抗性显著增强
为了探索ZmYUC2和ZmYUC4在玉米倒伏抗性方面的作用,发明人用动态根倒测定仪对抽雄期Zmyuc2/Zmyuc4双突变体及野生型对照材料(2021年廊坊)的根倒推力进行了测量,发现将Zmyuc4单突变体和Zmyuc2/Zmyuc4双突变体的茎秆基部推到同样角度(偏离垂直线的角度)所用的力,明显大于野生型对照材料,其中Zmyuc2/Zmyuc4双突变体所需力最大(图8A)。尤其值得注意的是,2021年廊坊7月初份廊坊发生暴风雨天气,田间ZC01(ZmYUC2和ZmYUC4基因编辑受体材料)背景的遗传材料发生不同程度的倒伏,而Zmyuc2/Zmyuc4的两个双突变体的倒伏率较野生型材料显著减轻(图8D);高密度种植是提高玉米单产的有效手段,但高密度种植会增加倒伏风险,降低玉米产量。为了检测Zmyuc突变体在不同种植密度下的根系抗倒伏能力,我们于2022年在廊坊进行了密度实验,分为三个密度D1(4.5万株/公顷)、D2(9万株/公顷)、 D3(13.5万株/公顷),每个密度3个重复。在吐丝期调查倒伏情况,倒伏度小于30°为Level 1,倒伏度小于60°但大于30°为Level 2,倒伏度大于60°为Level 3。对Zmyuc突变体和野生型(WT)的倒伏程度的测定表明,野生型(WT)、Zmyuc2和Zmyuc4的倒伏率随着种植密度的增加而增加,而Zmyuc2/4在3种密度条件下均保持直立(图8B和8C)。该实验表明Zmyuc4单突变体以及Zmyuc2/Zmyuc4双突变可用于玉米倒伏抗性的育种改良。
3.ZmYUC2和ZmYUC4基因的单突和双突均不会对玉米植株的地上部农艺性状带来不良影响
通过对2021年廊坊及2021年海南两个生长季的表型观察发现,Zmyuc2和Zmyuc4单突变体和Zmyuc2/4双突变体与对照材料之间的植株高度、叶片构型、雌雄花序(图9)、籽粒、果穗产量(图10)等相关性状没有明显的差异。报道显示,适当改变根系构型、不影响根系总量、不造成其余农艺性状较大改变方法,是培育高产抗倒伏玉米新品种的关键技术途径。该实验结果预示着ZmYUC2和ZmYUC4在玉米抗倒伏高产育种方面的巨大应用潜力。
实施例4 ZmYUC2和ZmYUC4基因通过调控根尖局部的生长素含量和分布参与调控根尖的向重力性
报道显示(Gallavotti A,Yang Y,Schmidt R J,et al.The relationship between auxin transport and maize branching[J].Plant physiology,2008,147(4):1913-1923.),DR5启动子(利用9个反向串联重复的生长素响应元件AuxRE创制的启动子)驱动报告基因表达的方法,如DR5::RFP,可以很好的反映生长素在植物体内的积累程度。
为确定ZmYUC2和ZmYUC4是否通过生长素丰度调控来影响玉米根的向重力性,发明人将DR5::RFP转基因材料与Zmyuc2和Zmyuc4的基因编辑突变体株系进行杂交,创制了Zmyuc2/DR5::RFP、Zmyuc4/DR5::RFP、Zmyuc2/Zmyuc4/DR5::RFP遗传材料,即将DR5::RFP导入到Zmyucm2、Zmyuc4单突变体,及Zmyuc2/Zmyuc4双突变体中。将根系沿重力方向培养,通过观察根尖局部的荧光强度所反映的生长素含量情况发现(图11A和图11C),ZmYUC2和ZmYUC4的基因突变后,根尖根冠部位(重力的感应组织)的生长素浓度明显降低,其中Zmyuc4单突变体根冠中的生长素含量降低较Zmyuc2单突体严重,而Zmyuc2/Zmyuc4双突变体中的生长素含量降低最严重;表明ZmYUC2和ZmYUC4基因可以调控根尖局部的生长素含量,也再次印证了ZmYUC2和ZmYUC4基因间存在功能冗余。
进一步的将根系沿垂直重力方向培养(模拟重力刺激),观察发现,在每种材料(野生型、Zmyuc2、Zmyuc4单突变体,及Zmyuc2/Zmyuc4双突变体)的根系伸长区,向地侧的生长素含量都较背地侧含量高(图11B和图11D)。而不同材料间比较发现,Zmyucm2、Zmyuc4单突变体,及Zmyuc2/Zmyuc4双突变体材料根系伸长区向地侧和背地侧的生长素含量都较野生型相应部位显著降低,其中Zmyuc2/Zmyuc4双突变体降低最厉害。比较向地侧和背地侧生长素所降低的程度还发现,Zmyucm2、Zmyuc4单突变体,及Zmyuc2/Zmyuc4双突变体材料根系伸长区向地侧和背地侧的生长素含量降低的程度(两侧的比率)都较野生型严重。受到重力刺激后,根系伸长区向地侧和背地侧的生长素含量差异是造成根系向重力响应和向重力弯曲的直接原因。因此,可以断定 ZmYUC2和ZmYUC4基因可通过调控根尖局部的生长素含量和分布参与根尖的向重力性调控。而根系响应重力刺激的敏感性和快慢决定了其向地弯曲的速度,进而决定了根茎夹角的大小。因此,ZmYUC2和ZmYUC4应该是通过调控根尖局部生长素的合成与含量,进而调控玉米根向重力性,进而调控玉米根茎夹角。
实施例5 ZmYUC2基因在现代玉米育种过程中受到了强的人工选择
发明人收集了350份中国和美国不同年代玉米育种材料。2021年冬天,在海南乐东试验基地对该350份玉米自交系材料的气生根夹角、层数、最上层气生根数目等表型进行了测量。通过比较不同年代材料间的性状变化规律发现,在中国特有的黄早四亚群材料中,玉米的气生根角度,在育种过程中经历了整体上由小向大的变化(早期材料的气生根夹角显著小于近现代材料),而气生根的数目和层数没有明显变化(图12A)。预示着气生根角度是黄早四亚群材料的重要育种靶标。进一步通过对350份材料进行平均13.4×的重测序,并利用XP-CLR的方法进行全基因组选择扫描,检测到了1,888个显著的选择区(selective sweeps)。其中,ZmYUC2基因落在了其中一个显著的的选择区中(图12B)。深入分析发现,ZmYUC2基因主要是在中国早期(20世纪60和70年代)到近现代(2000年至今)的育种过程中受到了显著的人工选择,并且主要在中国特有的黄早四亚群中受到选择。预示着ZmYUC2基因在中国玉米种质,尤其是黄早四亚群种质的育种改良过程中发挥着重要作用。倒伏抗性改良一直是玉米育种改良的首要育种目标,尤其是黄早四亚群种质改良的重要方向,这也从一个侧面反映了ZmYUC2基因的选择和应用对玉米抗倒伏育种的重要意义。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (16)
- 一种调控玉米根系夹角和倒伏抗性的基因,植株中所述基因突变后具有使玉米气生根生长角度变大和抗倒伏的表型,所述基因的多核苷酸序列选自下列组的序列之一:(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有使玉米气生根生长角度变大和抗倒伏的功能;(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列,该多核苷酸序列的突变具有使玉米气生根生长角度变大和抗倒伏的功能;或(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
- 表达盒、重组载体或细胞,所述表达盒、重组载体或细胞含有用于调控玉米根系夹角和倒伏抗性的基因序列,所述基因序列选自下列组的多核苷酸序列之一:(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有调控玉米根系夹角和倒伏抗性的功能;(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列;(e)具有(a)-(d)任一所示多核苷酸序列中300或500个以上连续多核苷酸序列的序列;或(f)与(a)-(e)之任一所述序列互补的多核苷酸序列。
- 一种基因突变体序列,由基因核苷酸序列突变获得,含有该基因突变体序列的玉米植株具有气生根生长角度变大和抗倒伏的表型,所述基因的核苷酸序列选自下列组的序列之一:(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,该多核苷酸序列的突变具有使玉米气生根生长角度变大和抗倒伏的功能;(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列;或(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
- 根据权利要求3所述的基因突变体序列,所述基因突变体序列通过突变的方式获得,所述突变包括在该基因的核苷酸序列上进行一个或多个核苷酸的取代、缺失和/或添加。
- 根据权利要求4所述的基因突变体序列,所述突变通过物理诱变、化学诱变、ZFN、TALEN和/或CRISPR/Cas基因编辑等技术获得。
- 根据权利要求5所述的基因突变体序列,其中所述的CRISPR/Cas基因编辑技术为CRISPR/Cas9,该技术所用的靶点序列选自下列组的序列之一:(a)序列为SEQ ID No:1、3、5、9或10所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段,其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸;或(b)与(a)所述的多核苷酸序列互补的核苷酸序列。
- 根据权利要求3-6之任一所述的基因突变体序列,所述基因突变体的核苷酸序列如SEQ ID No:11-14之任一所示。
- 一种增强玉米抗倒伏能力的方法,通过降低或抑制倒伏相关基因的正常表达或蛋白功能,使玉米植株增强抗倒伏能力,所述基因的多核苷酸序列选自下列组的序列之一:(a)如SEQ ID No:1、3、5、9或10所示的多核苷酸序列;(b)其编码氨基酸序列如SEQ ID No:2、4或6所示的多核苷酸序列;(c)在严谨杂交条件下能够与(a)或(b)中所述多核苷酸序列进行杂交的多核苷酸序列,抑制该多核苷酸序列的表达具有使玉米气生根生长角度变大和抗倒伏的功能;(d)与(a)-(c)任一所示的多核苷酸序列至少有95%或以上相似性的多核苷酸序列,抑制该多核苷酸序列的表达具有使玉米气生根生长角度变大和抗倒伏的功能;或(e)与(a)-(d)之任一所述序列互补的多核苷酸序列。
- 根据权利要求8所述的方法,其中所述的降低或抑制倒伏相关基因的正常表达或蛋白功能,包括通过RNA干扰和/或突变的方式获得。
- 根据权利要求9所述的方法,其中所述的降低或抑制基因表达或基因突变包括在该基因或基因启动子的核苷酸序列上进行一个或多个核苷酸的取代、缺失和/或添加。
- 根据权利要求8-10之任一所述的方法,其中所述的抑制抗倒伏基因正常表达的方式包括RNAi、物理诱变、化学诱变、ZFN、TALEN和/或CRISPR/Cas基因编辑等方法。
- 根据权利要求11所述的方法,其中所述的CRISPR/Cas为CRISPR/Cas9基因编辑方法,所用的靶点序列选自下列组的序列之一:(a)序列为SEQ ID No:1、3、5、7、8、9或10所示核苷酸序列中符合5’-Nx-NGG-3’序列排列规则的片段,其中N表示A、G、C和T中的任一种,14<X<30,且X为整数,Nx表示X个连续的核苷酸;或(b)与(a)所述的多核苷酸序列互补的核苷酸序列。
- 根据权利要求9-12之任一所述的方法,其中所述的基因突变后的核苷酸序列如SEQ ID No:11-14之任一所示。
- 一种非作为繁殖材料的植物细胞、组织、器官或产品,所述植物细胞、组织、器官或产品含有权利要求2所述的表达盒或权利要求3-7之任一所述的基因突变体序列。
- 权利要求1所述的基因、权利要求2所述的表达盒、重组载体或细胞、权利要求3-7之任一所述的基因突变体序列、权利要求8-13之任一所述的方法、及其获得的突变体材料或转化事件在育种中的应用。
- 根据权利要求15所述的应用,其中所述的在育种中的应用是指通过RNA干扰、基因突变、启动子突变和/或与突变体材料杂交的方式,使植株获得抗倒伏能力增强的表型。
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