阅读说明：本技术 一种抗虫低酚棉花品种的育种方法 (Breeding method of insect-resistant low-phenol cotton variety ) 是由 米换房 权月伟 唐光雷 翟雷霞 李继军 李文蕾 于 2019-10-25 设计创作，主要内容包括：本发明提供一种抗虫低酚棉花品种的育种方法,属于基因工程技术领域,包括对CAD1-A基因进行克隆,得CAD1-A-1质粒；构建CAD1-A基因的RNAi表达载体；采用根癌农杆菌介导法对所述转Bt基因抗虫棉的胚性愈伤组织进行遗传转化；其中,RNAi表达载体中CAD1-A基因由α-球蛋白启动子驱动,α-球蛋白启动子用Enh Ⅰ增强子进行修饰。该方法能够抑制Bt基因启动子及CAD1-A基因种子特殊启动子的甲基化,从而抑制复合转基因作物中Bt基因转录水平的显著降低,促进CAD1-A基因转录水平的降低；能够减少对胚性愈伤组织的毒害,抑制转化细胞的程序化死亡,增加转化频率。(The invention provides a breeding method of an insect-resistant low-phenol cotton variety, which belongs to the technical field of genetic engineering and comprises the steps of cloning a CAD1-A gene to obtain a CAD1-A-1 plasmid; constructing an RNAi expression vector of the CAD1-A gene; carrying out genetic transformation on the embryogenic callus of the Bt transgenic insect-resistant cotton by adopting an agrobacterium tumefaciens mediated method; wherein the CAD1-A gene in the RNAi expression vector is driven by the alpha-globin promoter, which is modified with the Enh I enhancer. The method can inhibit methylation of Bt gene promoters and CAD1-A gene seed special promoters, thereby inhibiting significant reduction of Bt gene transcription level in compound transgenic crops and promoting reduction of CAD1-A gene transcription level; can reduce the toxicity to the embryogenic callus, inhibit the programmed death of the transformed cells, and increase the transformation frequency.)

1. A breeding method of insect-resistant low-phenol cotton variety, which utilizes RNAi technology to silence cadinene synthetase gene CAD1-A in Bt transgenic insect-resistant cotton to prepare composite transgenic cotton variety, the concrete steps include:

s1, cloning the CAD1-A gene to obtain a CAD1-A-1 plasmid;

s2, constructing an RNAi expression vector of the CAD1-A gene;

s3, carrying out genetic transformation on the embryogenic callus of the Bt transgenic insect-resistant cotton by adopting an agrobacterium tumefaciens mediated method;

wherein the CAD1-A gene in the RNAi expression vector in step S2 is driven by an alpha-globulin promoter modified with an Enh I enhancer.

2. A breeding method according to claim 1, characterized in that: and constructing the RNAi expression vector of the CAD1-A gene by using the plant binary expression vector of the alpha-globulin promoter and the GUS reporter gene.

3. A breeding method according to claim 2, characterized in that: the construction method of the step S2 includes:

1) deleting part of GUS of the plant binary expression vector of the alpha-globulin promoter and GUS reporter gene to obtain a blank RNAi expression vector;

2) inserting a forward sequence of the CAD1-A gene into the vector obtained in the step 1) to obtain an RNAi expression vector of the CAD 1-A-R;

3) inserting the reverse sequence of the CAD1-A gene into the vector obtained in the step 2) to obtain the RNAi expression vector of the CAD1-A gene.

4. A breeding method according to claim 3, characterized in that: the method for preparing the hollow RNAi expression vector in the step 1) comprises the following steps:

a. carrying out PCR amplification on Intron1 of the Intron of the GUS gene, and recovering and purifying to obtain a target fragment I;

b. carrying out enzyme digestion on the plant binary expression vector by using SacI, and recovering and purifying to obtain a target fragment II;

c. carrying out enzyme digestion on the target fragment II by using XhoI, and recovering and purifying to obtain a target fragment III;

d. and connecting the target fragment I and the target fragment III to obtain the blank RNAi expression vector.

5. A breeding method according to claim 3, characterized in that: the preparation method of the CAD1-A gene forward sequence comprises the following steps: and carrying out PCR amplification on the CAD1-A-1 plasmid in the step S1 by taking SEQ ID NO.1 and SEQ ID NO.2 as primers, wherein the obtained PCR product is the forward sequence of the CAD1-A gene.

6. A breeding method according to claim 3, characterized in that: the preparation method of the CAD1-A gene reverse sequence comprises the following steps: and carrying out PCR amplification on the CAD1-A-1 plasmid in the step S1 by taking SEQ ID NO.3 and SEQ ID NO.4 as primers, wherein the obtained PCR product is the CAD1-A gene reverse sequence.

7. A breeding method according to claim 1, characterized in that: the embryogenic callus is pre-cultured for 12-18d, preferably 14-16d before genetic transformation.

8. A breeding method according to claim 1, characterized in that: the RNAi expression vector of the CAD1-A gene is transferred into the agrobacterium tumefaciens by electric shock transformation or heat shock method.

9. A breeding method according to claim 1, characterized in that: the specific step of genetic transformation in step S3 includes:

1) transformation, bacterial liquid activation and infection of agrobacterium tumefaciens: inoculating the embryogenic callus into a container, adding activated Agrobacterium tumefaciens bacterial liquid, infecting for 12-15min, continuously oscillating during the infection, discarding the bacterial liquid, repeatedly cleaning the embryogenic callus with sterile water, blotting, transferring into a co-culture medium containing ursolic acid monoester disodium phthalate and cyclodextrin glucose derivatives, and performing dark culture at 21-23 deg.C for 22-26 h;

2) screening culture, differentiation culture, rooting culture and seedling training.

10. Use of the breeding method as claimed in any one of claims 1 to 9 for breeding cotton varieties integrating grain, cotton and oil.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a breeding method of an insect-resistant low-phenol cotton variety.

Background

Gossypol is toxic to bacteria, fungi, insects, etc., and is an important component of cotton defense mechanisms. However, gossypol easily damages the gastric mucosa of humans and monogastric animals, causing disorders of digestive function, which has been a major cause of long-term impediments to better development and utilization of cotton seeds. Although gossypol can be removed by physical and chemical methods, the process is complex, the investment is large, the energy consumption is high, the cost is high, and the cottonseed cakes treated by the methods have different loss of nutrient components and lower edible safety. Therefore, breeding experts in all countries in the world develop the breeding work of the low-phenol cotton one after another, and hope that a new low-phenol cotton variety can be bred by means of genetic breeding, so that the cotton can produce not only fibers, but also cottonseed protein and cottonseed oil which can be directly utilized, and the new cotton-grain-oil three-in-one crop is formed. However, the low-phenol cotton variety obtained by conventional breeding has poor insect resistance due to no gossypol, is easily damaged by phytophagous pests and rats and rabbits, and is greatly limited in application and popularization. Therefore, the subsequent breeding target naturally concentrates on the variety breeding of cotton plants with high gossypol content and reduced gossypol content. Some researchers also try to improve the insect resistance of the low-phenol cotton by introducing exogenous Bt genes into the low-phenol cotton, but until now, the successful breeding report of the transgenic insect-resistant cotton with dominant low-phenol characters is not seen.

Disclosure of Invention

The invention aims to provide a breeding method of an insect-resistant low-phenol cotton variety, which can inhibit methylation of a Bt gene promoter and a CAD1-A gene seed special promoter, thereby inhibiting the obvious reduction of the Bt gene transcription level in compound transgenic crops and promoting the reduction of the CAD1-A gene transcription level; can reduce the toxicity to the embryogenic callus, inhibit the programmed death of the transformed cells, and increase the transformation frequency.

The technical scheme adopted by the invention for realizing the purpose is as follows:

provides a breeding method of insect-resistant low-phenol cotton varieties, which is characterized in that a composite transgenic cotton variety is prepared by silencing cadinene synthetase gene CAD1-A in transgenic Bt cotton through an RNAi technology, and the specific steps comprise:

s1, cloning the CAD1-A gene to obtain a CAD1-A-1 plasmid;

s2, constructing the RNAi expression vector of the CAD1-A gene;

s3, carrying out genetic transformation on the embryogenic callus of the Bt transgenic insect-resistant cotton by adopting an agrobacterium tumefaciens mediated method;

wherein the CAD1-A gene in the RNAi expression vector of step S2 is driven by an alpha-globulin promoter, and the alpha-globulin promoter is modified with an EnhI enhancer. The breeding method provided by the invention directly silences the cadinene synthetase gene CAD1-A in the transgenic Bt insect-resistant cotton by using RNAi technology, can obtain cotton varieties with good insect resistance, good quality of plants with phenol and cotton seeds without phenol, and is not limited by variety resources. In the composite transgenic plant, 67bp of homologous sequence of the transgenic promoter region is enough to cause gene silencing: homologous sequences of promoters of different genes close to each other are easy to combine and pair, and because two homologous sequences from different single strands are different in methylation degree, most of formed hybrid DNA double strands are in a hemimethylation state, so that a recognition site can be provided for DNA methyltransferase MET1 and a cofactor thereof in a promoter CpG region. By modifying the alpha-globulin promoter by using the Enh I enhancer, the homologous sequence of the promoter can be inhibited from being combined into a hybrid DNA double strand, and the methylation of the Bt gene promoter and the CAD1-A gene alpha-globulin promoter is inhibited, so that the obvious reduction of the Bt gene transcription level in the compound transgenic crops is inhibited, the expression of a CAD1-A RNAi vector is promoted, the reduction of the CAD1-A gene transcription level is promoted, the insect resistance of the compound transgenic plants is further improved, and the gossypol content of cotton seeds is reduced.

In some embodiments, the RNAi expression vector for the CAD1-a gene described above is constructed using the plant binary expression vector for the α -globulin promoter + GUS reporter gene described above.

In some embodiments, the constructing method of step S2 is:

deleting part of GUS of the plant binary expression vector of the alpha-globulin promoter and GUS reporter gene to obtain a blank RNAi expression vector;

inserting a CAD1-A gene forward sequence into the blank RNAi expression vector to obtain an RNAi expression vector of CAD 1-A-R;

and inserting the reverse sequence of the CAD1-A gene into the RNAi expression vector of the CAD1-A-R to obtain the RNAi expression vector of the CAD1-A gene.

In some embodiments, the method for preparing the blank RNAi expression vector comprises:

a. carrying out PCR amplification on Intron1 of the Intron of the GUS gene, and recovering and purifying to obtain a target fragment I;

b. carrying out enzyme digestion on the plant binary expression vector by using SacI, and recovering and purifying to obtain a target fragment II;

c. carrying out enzyme digestion on the target fragment II by using XhoI, and recovering and purifying to obtain a target fragment III;

d. and connecting the target fragment I and the target fragment III to obtain the blank RNAi expression vector.

In some embodiments, the CAD1-a gene forward sequence is prepared by: and (3) carrying out PCR amplification on the CAD1-A-1 plasmid in the step S1 by taking SEQ ID NO.1 and SEQ ID NO.2 as primers, wherein the obtained PCR product is the CAD1-A gene forward sequence.

In some embodiments, the reverse sequence of the CAD1-a gene is prepared by: and carrying out PCR amplification on the CAD1-A-1 plasmid in the step S1 by taking SEQ ID NO.3 and SEQ ID NO.4 as primers, wherein the obtained PCR product is the reverse sequence of the CAD1-A gene.

In some embodiments, the embryogenic callus is pre-cultured for 14-16d before genetic transformation. When the embryogenic callus is pre-cultured for 14-16d, the embryogenic callus is active in division state, is sensitive to agrobacterium and has high transformation rate.

In some embodiments, the RNAi expression vector of CAD1-A gene is transferred into the Agrobacterium tumefaciens by electric shock transformation or heat shock.

In some embodiments, the step of performing genetic transformation in step S3 comprises:

transformation, bacterial liquid activation and infection of agrobacterium tumefaciens: inoculating the embryogenic callus into a container, adding activated Agrobacterium tumefaciens bacterial liquid, infecting for 12-15min, continuously oscillating during the infection, discarding the bacterial liquid, repeatedly cleaning the embryogenic callus with sterile water, blotting, transferring into a co-culture medium containing ursolic acid monoester disodium phthalate and cyclodextrin glucose derivatives, and performing dark culture at 21-23 deg.C for 22-26 h;

screening culture, differentiation culture, rooting culture and seedling training. The agrobacterium is easy to breed and pollute, and can effectively inhibit overgrowth of the agrobacterium by co-culturing on a culture medium containing ursolic acid monoester disodium phthalate and cyclodextrin glucose derivatives, so that the concentration of the agrobacterium is kept at a proper concentration, the toxicity to embryonic callus is reduced, the programmed death of transformed cells is inhibited, and the transformation frequency is increased.

The invention also provides the application of the breeding method of the insect-resistant low-phenol cotton variety in cultivating the cotton variety integrating grain, cotton and oil.

The invention has the beneficial effects that:

1) the invention directly silences cadinene synthetase gene CAD1-A in the transgenic Bt insect-resistant cotton by RNAi technology, can obtain cotton varieties with good insect resistance, good quality of plants with phenol and cotton seeds without phenol, and is not limited by variety resources, compared with the traditional method of insect-resistant and low phenol fusion by breeding, the method has the advantages of short time consumption, less workload and simple and convenient operation, simultaneously improves the disease resistance, and can stably inherit;

2) according to the invention, the alpha-globulin promoter is modified by the Enh I enhancer, so that the homologous sequence of the promoter can be inhibited from being combined into a hybrid DNA double strand, and the methylation of the Bt gene promoter and the CAD1-A gene alpha-globulin promoter is inhibited, thereby inhibiting the obvious reduction of the Bt gene transcription level in the compound transgenic crops, promoting the expression of the CAD1-A RNAi vector, promoting the reduction of the CAD1-A gene transcription level, further improving the insect resistance of the compound transgenic plants and reducing the gossypol content of cotton seeds;

3) the invention can effectively inhibit the overgrowth of the agrobacterium by optimizing the co-culture medium, so that the concentration of the agrobacterium is kept at a proper concentration, the toxicity to the embryogenic callus is reduced, the programmed death of the transformed cells is inhibited, and the transformation frequency is increased.

Drawings

FIG. 1 shows the methylation rate of the 35S promoter in test example 1 of the present invention;

FIG. 2 shows the methylation rate of the α -globulin promoter in test example 1 of the present invention;

FIG. 3 shows the transcription level of CAD1-A gene in test example 2 of the present invention;

FIG. 4 is a graph showing the transcription level of the Cry1Ac gene in test example 2 of the present invention;

FIG. 5 shows browning rate, resistant callus rate and transformation rate in test example 3 of the present invention;

FIG. 6 shows the gossypol content and larval mortality in cotton seeds of test example 4 of the present invention.

Detailed Description

Unless otherwise indicated, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety as if set forth in their entirety.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any larger range limit or preferred value and any smaller range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is described, the described range should be construed as including ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. Where numerical ranges are described herein, unless otherwise stated, the stated ranges are intended to include the endpoints of the ranges and all integers and fractions within the ranges.

In addition, the words "a" and "an" preceding an element or component of the invention are intended to mean no limitation on the number of times that the element or component appears (i.e., occurs). Thus, "a" or "an" should be understood to include one or at least one and the singular forms of an element or component also include the plural unless the singular is explicitly stated.

Embodiments of the present invention, including embodiments of the invention described in the summary section and any other embodiments described herein below, can be combined arbitrarily.

The present invention is further described in detail with reference to the following examples: