阅读说明：本技术 一种高效获得籼稻赣香b转基因植株的农杆菌介导转化体系的方法 (Method for efficiently obtaining agrobacterium-mediated transformation system of transgenic plant of Ganxiang B of indica rice ) 是由 熊文涛 沈雨民 陈明亮 肖叶青 罗世友 熊焕金 吴小燕 于 2020-12-11 设计创作，主要内容包括：本发明公开了一种高效获得籼稻赣香B转基因植株的农杆菌介导转化体系的方法,包括以下步骤：受体材料的准备、农杆菌供体菌株培养、农杆菌侵染、抗性愈伤组织的筛选、分化培养基及抗性植株的检测。通过优化农杆菌转化时的菌液浓度、侵染时间、侵染条件、共培养条件等影响因素,建立了赣香B农杆菌介导转化体系。提高了现有农杆菌介导籼稻品种的转化效率,建立起了以胚性愈伤为受体材料的高效赣香B农杆菌转化体系。(The invention discloses a method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice, which comprises the following steps: preparing an acceptor material, culturing an agrobacterium donor strain, infecting agrobacterium, screening resistant callus, and detecting a differentiation culture medium and a resistant plant. The Ganxiang B agrobacterium-mediated transformation system is established by optimizing the influence factors such as the concentration of a bacterial liquid, the infection time, the infection conditions, the co-culture conditions and the like during agrobacterium transformation. Improves the transformation efficiency of the prior agrobacterium-mediated indica rice variety, and establishes a high-efficiency Ganxiang B agrobacterium transformation system taking embryogenic callus as a receptor material.)

1. A method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice comprises the steps of preparation of acceptor materials, culture of agrobacterium donor strains, agrobacterium infection, screening of resistant callus, differentiation culture medium and detection of the resistant plant, and is characterized in that:

the receptor material adopts the seed of Ganxiang B as an explant to induce callus, and embryogenic callus is obtained through induction and subculture;

connecting a target gene to an expression vector pCUbi1390 by a PCR amplification and enzyme digestion method, transferring the target gene into an escherichia coli DH5a body, extracting a plasmid, and then electrically exciting and transforming agrobacterium tumefaciens EHA 105;

activating the concentration of the agrobacterium liquid to OD₆₀₀=1.0, when infecting callus, the same volume of MS resuspends the concentration of agrobacterium liquid to OD₆₀₀=0.3, the MS resuspension contained acetosyringone at a concentration of 150 uM;

immersing the resuspended agrobacterium cells into rice callus, carrying out gentle culture on a shaking table adjusted to 50 rpm for 20-25 min, sucking redundant bacteria liquid on the callus by using sterile filter paper, transferring the callus onto a co-culture medium, carrying out co-culture for 48 h under the dark condition of 27 +/-1 ℃, washing the callus for 8-10 times by adding 250 mg/l cefotaxime sterile water once a small amount of agrobacterium appears on the callus, and fully sucking the callus by using sterile water filter paper after filtering;

transferring the sterilized filter paper to a screening culture medium after being sucked dry, carrying out dark culture at 27 +/-1 ℃ for 12 days, selecting normal callus after the first round of screening culture, transferring the normal callus to a new screening culture medium for second round of culture, selecting newly grown micro callus after the second round of screening culture for 10 days, transferring the newly grown micro callus to a new screening culture medium for third round of screening, and carrying out dark culture at 27 +/-1 ℃ for 5 days;

transferring the granular tiny calluses grown in the third round of screening culture medium to a differentiation culture medium, and culturing for 7 days at the temperature of 27 +/-1 ℃ in the dark; transferring the callus to a new differentiation culture medium, culturing for 4 days at the temperature of 27 +/-1 ℃ under the illumination condition, finally transferring the differentiated seedling to a rooting culture medium, and transplanting and hardening the seedling when the seedling grows to have a strong root system;

extracting the total DNA of each plant, and amplifying and detecting the hygromycin phosphotransferase gene in the plant by adopting a PCR method; if electrophoresis can detect the target band, the exogenous gene is integrated into the chromosome of the receptor cell.

2. The method for efficiently obtaining the agrobacterium-mediated transformation system of the transgenic plant of the Ganxiang B of the indica rice according to claim 1, which is characterized in that: the embryonic callus of gan Xiang B is obtained by the following induction method: taking mature Ganxiang B seeds, soaking the seeds in 70% ethanol for 1 min, treating the seeds with 50% sodium hypochlorite for 30 min, shaking the seeds on a shaking table adjusted to 180 rpm, washing the seeds with sterile water for 8 to 10 times, and finally putting the washed seeds on sterilized filter paper to dry the seeds for 5 min; putting seeds into a culture dish of an induction culture medium, and putting the culture dish into a dark environment at the temperature of 27 +/-1 ℃; after dark induction for 14 days, embryogenic callus is selected and put into a new induction culture medium for dark subculture for 4 days at the temperature of 27 +/-1 ℃.

3. The method for efficiently obtaining the agrobacterium-mediated transformation system of the transgenic plant of the Ganxiang B of the indica rice according to claim 2, which is characterized in that: the induction culture medium is MS culture medium, wherein 6-BA2.0 mg/L, NAA 0.3.3 mg/L, sucrose 30g/L and plant gel 0.4 g/L are added.

Technical Field

The invention relates to an agrobacterium-mediated transformation system, in particular to a method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice.

Background

Gan Xiang B is a three-line maintainer line bred by compound hybridization of 5 different rice maintainer lines from different sources, namely 03B, IR58025B, gold 23B, Xinlu B, Jiangnong Zao No. 2B and the like in the research laboratory. The stems are thick and strong, the individual advantages are strong, and the number of main stem leaves is 12-13%; the functional leaves in the mature period grow vigorously, are not easy to senilism and have good color loss; the plant type is moderate in compactness and scattering, the seedlings are lower, the tillering capability is strong, the root system is developed, the sword leaves are upright, the big ears and the big particles are dense in seed setting, the ear length is about 22 cm, the average number of the particles per ear is 150, the thousand-particle weight is 28-29g, the particles are not easy to fall, the length-width ratio is 3.3, and the fragrance is achieved; the stigma exsertion rate is high, and the amylose content is high.

Preliminary detection ofPib、Pikm、Pi33、Pi5The rice blast resistance genes are distributed on chromosomes 2, 8, 9 and 11 respectively. Detection of phosphorus-containing high-efficiency genesPup1The functional indicia of (1). Has early maturity dominance, is suitable for being matched with indica-japonica hybrid combination, and has high seed production yield. There have been 15 combinations approved by provincial level in Fujian, Hainan, Yunnan, Sichuan, Chongqing, Guizhou, Zhejiang and Jiangxi provinces and cities for 19 times respectively: gangyou Ming (Jian examined rice 2010012, Min examined rice 2011004, Dian examined rice 2012017, Yu examined rice 2014004), Gangyou 607 (Jian examined rice 2012007), Gangxiangyou 702 (Chuan examined rice 2013017), Gangyou 5359 (Qian examined rice 2013003), Gangyou 810 (Min examined rice 2014008, Lu super rice 201914), Gangxiangyou 510 (Chuan examined rice 2014018), Gangyou 671 (Min examined rice 2015003), Gangyou 673 (Min examined rice 2015010), Gangyou 9141 (Zhengexamined rice 2015008), Gangyou 676 (Min examined rice 2016003), Gangyou 157 (Jian examined rice 2016006), Gangxiangyou (gan examined rice 20170027), Gangxiangyou 993 (Gangju examined rice 20170024), Gangyou 7076 (Dian examined rice 2017009), and Gangyou 9812 (Jian examined rice 9812)Audited rice 2017003, audited rice 20180025).

Constructing a near isogenic introduction line of Ganxiang B; constructing 180 strains of introgression line group BC2F8 with a maintainer line Ganxiang B as a recurrent parent and Dongxiang wild rice as a donor parent, obtaining two cold-resistant strains by field low-temperature screening, and subsequently performing expression spectrum difference sequencing on the Ganxiang B and the cold-resistant strains at normal temperature and low temperature, analyzing and comparing the difference between the two strains, finding out related cold stress genes and knowing the molecular regulation mechanism of cold resistance.

In order to verify the biological functions of the screened cold stress genes, an over-expression vector of the genes is constructed and then transferred into the embryogenic callus of Ganxiang B of indica rice. As is well known, the transformation efficiency of most indica rice is extremely low, and in order to better research the biological function of related genes in the Ganxiang B, the research on an agrobacterium-mediated transformation system for efficiently obtaining transgenic plants of the Ganxiang B of the indica rice is necessary. The specific conditions and methods for genetic transformation of different plants by using Agrobacterium vary depending on the species. At present, no agrobacterium-mediated transformation system for efficiently obtaining transgenic plants of the Jiangxiang B of the indica rice exists.

Disclosure of Invention

The invention aims to provide a method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice, which establishes the Ganxiang B agrobacterium-mediated transformation system by inducing and culturing callus of Ganxiang B, taking agrobacterium tumefaciens as a medium and optimizing influence factors such as bacterial liquid concentration, infection time, infection conditions, co-culture conditions and the like during agrobacterium transformation, thereby obtaining the transgenic plant of Ganxiang B. Provides powerful technical support for the research of functional genes in Ganxiang B in the future.

The purpose of the invention is realized as follows:

a method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice comprises the steps of preparation of acceptor materials, construction of transformation vectors, culture of agrobacterium donor strains, agrobacterium infection, screening of resistant callus, detection of a differentiation culture medium and a resistant plant and the like, and is characterized in that:

(1) the receptor material adopts the seed of Ganxiang B as an explant to induce callus, and embryogenic callus is obtained through induction and subculture;

(2) connecting a target gene to an expression vector pCUbi1390 by a PCR amplification and enzyme digestion method, transferring the target gene into an escherichia coli DH5a body, extracting a plasmid, and then electrically exciting and transforming agrobacterium tumefaciens EHA 105;

(3) activating the concentration of the agrobacterium liquid to OD₆₀₀=1.0, when infecting callus, the same volume of MS resuspends the concentration of agrobacterium liquid to OD₆₀₀=0.3, the MS resuspension contained acetosyringone at a concentration of 150 uM;

(4) immersing the resuspended Agrobacterium cells in the rice callus, gently culturing for 20-25 min on a shaker adjusted to 50 rpm, and sucking the excess bacterial liquid on the callus with sterile filter paper. These calli were then transferred to co-culture medium and co-cultured for 48 h at 27. + -. 1 ℃ in the dark. Once a small amount of agrobacterium is generated in the callus, the callus is washed for 8 to 10 times by adding 250 mg/l cefotaxime sterile water, and the callus is fully sucked and dried by sterile water filter paper after being filtered;

(5) after the filter paper is sterilized and sucked dry, the filter paper is transferred to a screening culture medium and cultured in the dark at the temperature of 27 +/-1 ℃ for 12 days. After the first round of screening culture, normal callus is selected and transferred to a new screening culture medium for the second round of culture. After 10 days of second round screening culture, selecting newly grown tiny callus, transferring the tiny callus to a new screening culture medium for third round screening, and carrying out dark culture at 27 +/-1 ℃ for 5 days;

(6) transferring the granular tiny calluses grown in the third round of screening culture medium to a differentiation culture medium, and culturing for 7 days at the temperature of 27 +/-1 ℃ in the dark; transferring the callus to a new differentiation culture medium, culturing for 4 days at the temperature of 27 +/-1 ℃ under the illumination condition, finally transferring the differentiated seedling to a rooting culture medium, and transplanting and hardening the seedling when the seedling grows to have a strong root system;

(7) extracting the total DNA of each plant, and amplifying and detecting the hygromycin phosphotransferase gene in the plant by adopting a PCR method; if electrophoresis can detect the target band, the exogenous gene is integrated into the chromosome of the receptor cell.

Further, the Ganxiang B embryogenic callus is obtained by the following induction method: taking Ganxiang B mature seeds, firstly soaking the seeds in 70% ethanol for 1 min, then treating the seeds with 50% sodium hypochlorite for 30 min, shaking the seeds on a shaking table adjusted to 180 rpm, washing the seeds with sterile water for 8-10 times, and finally putting the washed seeds on sterilized filter paper and airing the seeds for 5 min. Putting seeds into a culture dish of an induction culture medium, and putting the culture dish into a dark environment at the temperature of 27 +/-1 ℃; after dark induction for 14 days, embryogenic callus is selected and put into a new induction culture medium for dark subculture for 4 days at the temperature of 27 +/-1 ℃.

Further, the induction culture medium is MS culture medium, wherein 6-BA2.0 mg/L, NAA 0.3.3 mg/L, sucrose 30g/L and plant gel 0.4 g/L are added.

The invention successfully obtains the transgenic plant of the gan flavor B by constructing the agrobacterium-mediated transformation system of the gan flavor B, and the transformation efficiency is as high as 16.4 percent and is improved by nearly 1 time compared with the transformation efficiency of the common indica rice variety. Most importantly, provides powerful technical support for the research on the related functional genes in the Ganxiang B.

Drawings

FIG. 1 is the structural diagram of the Ganxiang B induced callus of the present invention;

FIG. 2 is a diagram of the resistant callus after conversion of gan Xiang B of the present invention;

FIG. 3 is the diagram of the resistant seedlings regenerated on the differentiating culture medium of Jiangxiang B of the present invention;

FIG. 4 is the field planting map of the transgenic seedling of Jiangxiang B of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples.

The invention comprises a method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice, which mainly comprises the steps of adopting a mature seed of Ganxiang B as an explant to induce callus, obtaining embryogenic callus through culture, introducing pCUbi1390 plasmid with hygromycin resistance into the Ganxiang B callus through agrobacterium tumefaciens mediated transformation, obtaining a sterile seedling of Ganxiang B through screening and differential culture of the resistant callus, and detecting the integration condition of exogenous hygromycin phosphotransferase genes through PCR (polymerase chain reaction) to obtain the transgenic plant of Ganxiang B.

A method for efficiently obtaining an agrobacterium-mediated transformation system of a transgenic plant of Ganxiang B of indica rice comprises the steps of preparation of acceptor materials, construction of transformation vectors, culture of agrobacterium donor strains, agrobacterium infection, screening of resistant callus, and detection of a differentiation culture medium and a resistant plant, and comprises the following steps:

(1) the receptor material adopts the seed of Ganxiang B as an explant to induce callus, and embryogenic callus is obtained through induction and subculture;

(2) connecting a target gene to an expression vector pCUbi1390 by a PCR amplification and enzyme digestion method, transferring the target gene into an escherichia coli DH5a body, extracting a plasmid, and then electrically exciting and transforming agrobacterium tumefaciens EHA 105;

(3) activating the concentration of the agrobacterium liquid to OD₆₀₀=1.0, when infecting callus, the same volume of MS resuspends the concentration of agrobacterium liquid to OD₆₀₀=0.3, the MS resuspension contained acetosyringone at a concentration of 150 uM;

(4) immersing the resuspended Agrobacterium cells in the rice callus, gently culturing for 20-25 min on a shaker adjusted to 50 rpm, and sucking the excess bacterial liquid on the callus with sterile filter paper. These calli were then transferred to co-culture medium and co-cultured for 48 h at 27. + -. 1 ℃ in the dark. Once a small amount of agrobacterium is generated in the callus, the callus is washed for 8 to 10 times by adding 250 mg/l cefotaxime sterile water, and the callus is fully sucked and dried by sterile water filter paper after being filtered;

(5) after the filter paper is sterilized and sucked dry, the filter paper is transferred to a screening culture medium and cultured in the dark at the temperature of 27 +/-1 ℃ for 12 days. After the first round of screening culture, normal callus is selected and transferred to a new screening culture medium for the second round of culture. After 10 days of second round screening culture, selecting newly grown tiny callus, transferring the tiny callus to a new screening culture medium for third round screening, and carrying out dark culture at 27 +/-1 ℃ for 5 days;

(6) transferring the granular tiny calluses grown in the third round of screening culture medium to a differentiation culture medium, and culturing for 7 days at the temperature of 27 +/-1 ℃ in the dark; transferring the callus to a new differentiation culture medium, culturing for 4 days at the temperature of 27 +/-1 ℃ under the illumination condition, finally transferring the differentiated seedling to a rooting culture medium, and transplanting and hardening the seedling when the seedling grows to have a strong root system;

(7) extracting the total DNA of each plant, and amplifying and detecting the hygromycin phosphotransferase gene in the plant by adopting a PCR method; if electrophoresis can detect the target band, the exogenous gene is integrated into the chromosome of the receptor cell.

Example (b):

1. inducing the embryonic callus of Ganxiang B;

taking Ganxiang B mature seeds, and carrying out artificial shelling and disinfection on the seeds, wherein the procedures are as follows: soaking with 70% ethanol for 1 min, treating with 50% sodium hypochlorite for 30 min, and shaking on a shaker adjusted to 180 rpm. Washing with sterile water for 8-10 times, and air drying the washed seeds for 5 min. 12-13 seeds are placed in each culture dish poured with an induction culture medium (MS +6-BA 2.0 mg/L + NAA 0.3 mg/L + sucrose 30g/L + plant gel 0.4 g/L) and cultured in dark at the temperature of 27 +/-1 ℃. After dark induction for 14 days, embryogenic callus is selected, evenly divided into 3 equal parts and put into a new induction medium for dark subculture for 4 days at the temperature of 27 +/-1 ℃.

2. Introduction of pCUbi1390 plasmid into Ganxiang B callus

Using cDNA extracted from the material with higher light as template, and performing PCR amplification with Os05g15770FKpnI 5'-GGGGTACCATGGCGTCCCGACGCCTTG-3', Os05g15770RSpeI 5'-GACTAGTTCACAGAACCTGATCCAGGAGACC-3' primer and KODFX enzyme (Takara)OsHI-XIPA CDS sequence. By usingKpnI andSpei enzyme digestion PCR purified product and pCUbi1390 expression vector, then are connected by DNA ligase, are transferred into colon bacillus DH5a, and correct is obtained by screening clone, enzyme digestion detection and sequencing analysisOsHI-XIPAnd (3) performing electric excitation on the overexpression vector to transform the agrobacterium EHA105, and finally co-culturing the agrobacterium EHA105 with the target plasmid and the callus.

3. Agrobacterium-mediated transformation System establishment

Picking from solid-state marking platesAgrobacterium EHA105 monoclonal strain, placed in a 50 ml centrifuge tube containing 5 ml YEB broth. The tube was placed on a shaker set at 200 rpm and cultured in the dark at 28 ℃ for 20 to 24 hours, and then the culture solution in the tube was transferred to a 500 ml Erlenmeyer flask containing 100 ml YEB and cultured under the same culture conditions. When the product is O.D.₆₀₀When reaching 1.0, the agrobacterial cells are enriched by centrifugation at 8000 x g for 15 min at 4 ℃, and finally the agrobacterial cells are resuspended in suspension medium. Immersing the resuspended Agrobacterium cells in the rice callus, gently culturing for 20-25 min on a shaker adjusted to 50 rpm, and sucking the excess bacterial liquid on the callus with sterile filter paper. These calli were then transferred to co-culture medium and co-cultured for 48 h at 27. + -. 1 ℃ in the dark. Once a small amount of agrobacterium is appeared in the callus, the callus is washed 8 to 10 times by adding cefotaxime sterile water with proper concentration, and the callus is transferred to a screening culture medium after being sucked dry by a sterilizing filter paper and cultured in dark at the temperature of 27 +/-1 ℃ for 12 days. After the first round of screening culture, normal callus is selected and transferred to a new screening culture medium for the second round of culture. After the second round of screening culture for 10 days, selecting new growing tiny callus to transfer to new screening culture medium for the third round of screening, and culturing in dark at 27 +/-1 ℃ for 5 days. Transferring the granular tiny calluses grown in the third round of screening culture medium to a differentiation culture medium, and culturing for 7 days at the temperature of 27 +/-1 ℃ in the dark; the calli were then transferred to fresh differentiation medium and cultured for 4 days at 27 + -1 deg.C in light. And finally transferring the differentiated seedlings to a rooting culture medium, and transplanting and hardening the seedlings when the seedlings grow strong and have developed root systems.

4. Detection of transgenic plants

Shearing young leaves of the plant, extracting DNA by referring to a CTAB method of Wangchun and the like, and amplifying and detecting a Hygromycin (HPT) gene in the plant by adopting a PCR method. Primer: hpt 557-F5'-ACACTACATGGCGTGATTTCAT-3', hpt 557-R5'-TCCACTATCGGCGAGTACTTCT-3'. Reaction system (10 ul system): 30-50 ng of DNA template, 1.1 XT 3 Super PCR Mix 9.1. mu.l, 10. mu.M primers 0.2. mu.L each. Reaction conditions are as follows: 2 min at 98 ℃, 10 s at 54 ℃, 10 s at 72 ℃, 32 Cycles, 2 min at 72 ℃ and 1 min at 25 ℃.