CN109971772B - Breeding method of low-temperature-resistant cotton variety - Google Patents
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Abstract
The invention discloses application of spinacia oleracea choline monooxygenase gene (AhCMO for short) in cultivating low-temperature-resistant plant varieties, and application of expression vectors and plant cells containing the AhCMO gene in cultivating low-temperature-resistant plant varieties. The invention also discloses a breeding method of the low temperature resistant cotton variety, namely, the AhCMO gene is transformed into the cotton variety, and a material with high AhCMO gene expression level is selected. The AhCMO gene is used for low temperature resistant breeding of plants, and a new field is developed for the application of the gene; secondly, the AhCMO transgenic cotton overcomes the sensitivity of cotton to low temperature, not only avoids the reduction of cotton yield and the reduction of fiber quality, but also can enlarge the cotton planting area and improve the income of farmers.
Description
Technical Field
The invention belongs to the field of cotton breeding methods, and particularly relates to application of spinacia oleracea choline monooxygenase genes in cultivation of low-temperature-resistant cotton varieties; and the application of the expression vector or plant cell containing the gene in culturing low temperature resistant cotton variety; also relates to a breeding method of the low temperature resistant cotton variety.
Background
Cotton (GossypumiumhirsutumL.) originates in tropical and subtropical regions, and is therefore very sensitive to cold stress. Cold stress significantly inhibits cotton seed germination and seedling growth, and also causes failure in fertilization, boll shedding and even plant death, thereby causing a decrease in cotton yield and fiber quality. The cotton planting of China is mainly distributed in the northwest inland of Xinjiang and other cold areas of high latitude, high altitude and other cold areas, cold stress becomes a main limiting factor of cotton production of China, and cotton linter yield loss caused by the cold stress can reach 30% -40%, so the cold stress becomes an urgent problem to be solved in the cotton production of China.
The rapid synthesis and accumulation of large amounts of small molecule compounds to maintain the normal function of cells is an adaptive response of plants to the outside world when subjected to saline and alkaline or other environmental stresses. Betaine belongs to one of the important small molecular compounds, and under the condition of salt stress, a large amount of betaine is accumulated in cells of many plants, particularly Li family and gramineae plants, so that the cell osmotic regulation capacity is improved, and the structures and functions of intracellular macromolecular proteins and biological membranes are stabilized. In higher plants, betaine synthesis is initiated with Choline and is accomplished by a two-step catalytic reaction, the enzyme catalyzing the first step is Choline Monooxygenase (CMO), which is the rate-limiting enzyme in the process of betaine biosynthesis. By increasing the expression level of the betaine in the plant, the salt tolerance or drought resistance of the plant can be improved. Currently, the gene has been isolated from plants such as the Chenopodiaceae spinach (Rathinasabaphathi et al PNAS,1997,94(7):3454-3458), beet (Russell et al Plant Physiology,1998,116: 859-.
Spinacia oleracea (also known as spinacia oleracea or Atriplex hortensis) belongs to genus Atriplex of family Chenopodiaceae, is a betaine natural storage plant, and is resistant to severe conditions such as cold, drought, and high salinity (Tao et al, Scientific Reports,2018,8(1): 2707; Shen et al, Theoretical and Applied Genetics, 2002). At present, the CMO gene (abbreviated as AhCMO) of spinacia oleracea has been cloned (Shen, et al, BioEngineers, 2001,1: 1-6). Through constructing an overexpression vector (pBI121-AhCMO), AhCMO gene is transformed into tobacco and cotton to obtain AhCMO gene transformed tobacco and cotton, and experiments prove that the introduced AhCMO gene improves the drought tolerance and the saline-alkali tolerance of the tobacco (Dianthus hai-red. Master thesis of Chinese academy of agricultural sciences, 2015).
Through retrieval, no report about the application of the AhCMO gene in cotton low temperature resistant breeding is found.
Disclosure of Invention
In order to solve the problem of low-temperature cold injury of plants, the invention aims to provide application of spinacia oleracea choline monooxygenase gene (AhCMO) in cultivation of low-temperature-resistant plant varieties.
In the application, the spinacia oleracea choline monooxygenase gene (abbreviated as AhCMO) consists of a nucleotide sequence shown in SEQ ID NO: 1.
In the application, the encoding protein of the spinacia oleracea choline monooxygenase gene (AhCMO) consists of an amino acid sequence shown in SEQ ID NO: 2.
The plants in the application are cotton, corn, rice or soybean and the like.
The invention also aims to provide application of the expression vector containing the spinacia oleracea choline monooxygenase gene (AhCMO) in cultivation of low-temperature-resistant plant varieties.
In the application, the expression vector refers to a plant expression vector PBI121 and the like.
In the application, the plant refers to cotton, corn, rice or soybean and the like.
The third purpose of the invention is to provide the application of the plant cell containing the spinacia oleracea choline monooxygenase gene (AhCMO) in the cultivation of low-temperature resistant plant varieties.
In the application, the plant refers to cotton, corn, rice or soybean and the like.
The fourth purpose of the invention is to provide a method for cultivating a low-temperature-resistant cotton variety, which comprises the step of transforming spinacia oleracea choline monooxygenase gene (AhCMO) into the cotton variety by a transgenic method.
The method for cultivating the low-temperature-resistant cotton variety comprises the following specific steps:
(1) cloning genes: performing PCR amplification by using AhCMO-F and AhCMO-R as primers and spinacia oleracea cDNA as a substrate to obtain a PCR amplification product, namely an AhCMO gene fragment; wherein the primer is as follows:
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GTGTTGCTTTTGATGCTTTCGTAAGCGATCCATTC-5’；
(2) constructing an intermediate vector: carrying out double digestion on the AhCMO gene fragment obtained in the step (1) by using PstI and XhoI, and recovering the gene fragment after agarose gel electrophoresis; the vector pUC19 was digested with PstI and XhoI, the linear pUC19 fragment was recovered, AhCMO was ligated into pUC19 containing the expression-enhancing sequences of Cozk, omega and p1oyA, and the genes were ligated with CaMV35 promoter and Nos terminator to construct an intermediate vector pUC 19-AhCMO;
(3) construction of an expression vector: carrying out double digestion on the intermediate vector pUC19-AhCMO obtained in the step (2) by EcoRI and Hihd III, and recovering a fragment containing the AhCMO gene; respectively connecting the EcoRI and Hihd III double-enzyme-digested vector pBI121 to construct an over-expression vector pBI 121-AhCMO;
(4) and (3) transformation: taking 100 mu L of prepared agrobacterium GV3101 competent cells, adding 10 mu g of pBI121-AhCMO plasmid DNA, mixing uniformly, and placing on ice for 5 min; then adding the competence mixed solution into a 2mm electric shock cup, performing electric shock at 2500V, quickly adding 800 mu L YEB liquid culture medium after electric shock, and performing induced culture for 5h at 28 ℃ and 180 rpm; taking out the induced bacterial liquid, coating the bacterial liquid on YEB plates containing 50 mu g/mL of kanamycin and rifampicin respectively, and culturing the bacterial liquid at the temperature of 28 ℃ for 48 hours; selecting a single clone, inoculating the single clone into 5mL YEB liquid culture medium containing kanamycin and rifampicin (the concentration is 50 mu g/mL), shaking the bacteria for 36 hours under the condition of 180rpm, and performing small-amount plasmid digestion and PCR identification; transforming the constructed AhCMO vector into cotton by utilizing an agrobacterium infection method to obtain an AhCMO transgenic cotton regenerated seedling;
(5) identification and screening: performing RT-PCR identification on the transgenic regenerated seedlings obtained in the step (4); harvesting seeds of T0 generation identified as positive plants; sowing the harvested transgenic T0-generation seeds, extracting DNA from well-grown T1-generation leaf tissues, carrying out RT-PCR detection, screening positive materials, continuously culturing, selecting strains with high AhCMO gene expression level, and breeding into AhCMO transgenic cotton varieties.
The fifth purpose of the invention is to provide a breeding method for cultivating low-temperature resistant cotton varieties by using transgenic cotton containing AhCMO genes, wherein the breeding method is a backcross breeding method or a cross breeding method;
the backcross breeding method comprises the following steps:
(1) taking transgenic cotton containing AhCMO gene as non-recurrent parent, taking cotton variety with excellent character as recurrent parent, and crossing to obtain F1Generation;
(2) with F1Backcrossing with the cotton variety in step (1) as male parent to obtain BC1F1Generation; taking single seed as unit, extracting BC1F1Single grain genome DNA is substituted, PCR identification is carried out by taking the genome DNA as a substrate and AhCMO-F and AhCMO-R as primers, and a descendant with the size of 1314bp of PCR amplification product is selected; backcrossing with the cotton variety of step (1) as female parent to obtain BC2F1Generation; wherein the primer sequence is as follows:
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’；
(3) repeating the backcrossing in the step (2) for 3-6 times; and (3) finally selfing for 1 generation, performing PCR identification on the seeds of selfed progeny in the step (2), and keeping the AhCMO gene in the identified single-plant progeny not to be separated, namely, breeding a new variety containing the AhCMO gene.
The transgenic cotton containing the AhCMO gene in the step (1) of the backcross breeding method is CMO24 or a CMO24 derivative line containing the AhCMO gene.
The cotton variety with excellent properties in the step (1) of the backcross breeding method is a cotton variety popularized and applied in production; or a newly developed cotton line. Wherein the characters refer to agronomic characters, fiber quality and the like.
The PCR reaction system (20ul) described in step (2) of the backcross breeding method described above: 1 mul of transgenic cotton genome DNA to be detected, 1 mul of each primer, and Mix10 mul are added with sterile water to 20 mul; the reaction conditions of the PCR are as follows: 94 ℃ for 3 min; 94 ℃,30 Sec; 56 ℃,30 Sec; 72 ℃,30 Sec; circulating for 30 times; 72 ℃ for 10 min.
The cross breeding method comprises the following steps:
(1) taking transgenic cotton containing AhCMO gene as one of parents, taking a good cotton variety as the other parent for hybridization to obtain F1Generation;
(2) and obtaining F1Selfing and harvesting to obtain F2Generation;
(3) identification and selection of F by molecular marker assistance2Planting single grains with AhCMO gene in the generation to identify positive progeny, and selecting plants for selfing; repeating the PCR identification and the plant selection for selfing for 4-6 generations to breed a new cotton variety containing the AhCMO gene; the molecular marker is obtained by performing PCR amplification by using genome DNA of cotton grains to be detected as a substrate and AhCMO-F and AhCMO-R as primers, and selecting a progeny material with an amplification product size of 1314 bp.
The cotton containing the AhCMO gene in the step (1) of the cross breeding method is CMO 24. Accession number of CMO 24: CGMCC No. 17381.
Sources of CMO 24: the AhCMO gene is transformed into a cotton line R15(R15 is a cotton line selected from cotton variety koji 312 (or Coker312) by the institute of biotechnology of Chinese academy of agricultural sciences), and then a line with the highest AhCMO gene expression level and strong low-temperature resistance is selected from the transformants and named as CMO 24.
The invention also provides a molecular marker for identifying AhCMO transgenic cotton, and the molecular marker consists of a nucleotide sequence shown in SEQ ID NO. 1.
The invention also provides a primer pair for amplifying the molecular marker, wherein the primer pair consists of AhCMO-F and AhCMO-R;
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’。
the invention also provides a detection kit of AhCMO transgenic cotton, wherein a primer pair in the detection kit consists of AhCMO-F and AhCMO-R;
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’。
compared with the prior art, the invention has the advantages and beneficial effects that: (1) the spinacia oleracea choline monooxygenase (AhCMO) gene is used for low temperature resistant breeding of plants for the first time, a good gene source is provided for low temperature resistant breeding of the plants, and a new field is developed for application of the AhCMO gene; (2) the AhCMO gene in the AhCMO transgenic cotton strain obtained by the invention has high expression quantity and strong low temperature resistance, and provides a new way for low temperature resistant breeding of plants; (3) the cotton variety bred by the method has strong low temperature resistance, overcomes the sensitivity of the cotton to low temperature, can overcome the cold damage caused by low temperature, avoids the reduction of yield and fiber quality, can enlarge the planting area and enlarge the planting area, or can advance the seeding period and prolong the growth period, thereby improving the yield and the fiber quality and increasing the income of farmers.
And (3) biological preservation: the invention relates to upland cotton (Gossypiumhirsutum Linn) CMO24, which is a transgenic cotton line bred by transforming spinacia spinaches choline monooxygenase gene (AhCMO) into cotton variety R15(R15 is a cotton line selected from cotton variety Korea 312 (or Coker312) by the institute of biotechnology, Chinese academy of agricultural sciences) through a transgenic method and selecting a material with high AhCMO gene expression and strong low-temperature resistance from the obtained transformant. CMO24 has been deposited in the China general microbiological culture Collection center at 25.2.2019 under the following deposition numbers: CGMCC No. 17381.
Drawings
FIG. 1 is a schematic diagram of AhCMO gene expression vector pBI 121-AhCMO.
FIG. 2 is a PCR identification electropherogram of AhCMO gene; wherein CK is wild type R15, and 1-23 are positive identification results of 23 transgenic plants respectively.
FIG. 3 shows the expression level of AhCMO gene in 6-week-old transgenic cotton plants; wherein 1 is wild type R15, 2 is CMO20, and 3 is CMO 24.
FIG. 4 is a photograph of seedlings of wild type R15 and transgenic cotton line CMO24 before and after cryo-treatment; wherein 1 is wild type R15 before treatment, and 2 is CMO24 before treatment; 3 is treated wild type R15 and 4 is treated CMO 24.
FIG. 5 is a photograph of a blue-stained portion of a wild type R15 and a transgenic cotton line CMO24 leaf discs before and after cryogenic treatment; wherein 1 is wild type R15 before treatment, 2 is CMO24 before treatment, 3 is wild type R15 after treatment, and 4 is CMO24 after treatment.
FIG. 6 is a bar graph of chlorophyll content in leaves of wild type R15 and transgenic cotton line CMO24 before and after cryo-treatment; wherein 1 is wild type R15, and 2 is CMO 24.
FIG. 7 is a bar graph of the free nitrogen content of leaves from wild type R15 and transgenic cotton line CMO24 cotton before and after cryo-treatment; wherein 1 is wild type R15, and 2 is CMO 24.
FIG. 8 is a bar graph of betaine content in leaves of wild type R15 and transgenic cotton line CMO24 before and after cryo-treatment; wherein 1 is wild type R15, and 2 is CMO 24.
Detailed Description
The invention is further illustrated and described by the following examples, which do not limit the scope of the invention in any way. Unless otherwise specified, the reagents used in the following examples are conventional reagents, and the methods used are conventional in the art, or according to the instructions for the reagents purchased.
Example 1 cloning of AhCMO Gene and construction of expression vector
Materials and methods
1) Cotton material: the cotton strain R15 is a self-bred strain of the institute of biotechnology, Chinese academy of agricultural sciences, and is stored by the institute of molecular Breeding of crops, institute of biotechnology, Chinese academy of agricultural sciences.
2) Strain: escherichia coli Transl-T1, Agrobacterium GV 3101.
3) Carrier: pEASY-Blunt, pEASY-T5, pUC19, pBI 121.
4) Tool and modifying enzymes: various restriction enzymes and modified enzymes were purchased from TaKaRa, NEB and TIANGEN.
5) Chemical reagents: the chemicals are all analytically pure at home and abroad.
6) Primer synthesis: synthesized by Shanghai Biotechnology service, Inc.
7) Sequencing: completed by Huada Gene Co.
(II) test procedure
The AhCMO gene is connected into a pBI121 vector to construct a single gene expression vector, and the operation steps are as follows:
1) primers were designed based on degenerate codons, and the full length of the AhCMO coding region cDNA gene was amplified, the gene fragment was cleaved with PstI and XhoI, and the gene AhCMO was recovered after agarose gel electrophoresis (see Dianthus hai red, the Master thesis of Chinese academy of agricultural sciences 2015), and the AhCMO gene was sequenced to consist of the nucleotide sequence shown in SEQ ID NO: 1. The protein coded by the AhCMO gene consists of an amino acid sequence shown in SEQ ID NO. 2.
2) The vector pUC19 was digested with PstI and XhoI to recover a linear vector fragment, and the AhCMO gene obtained in step 1) was ligated to pUC19 containing an expression-enhancing sequence such as Cozk, omega and p1oyA, and the CaMV35s promoter and Nos terminator were ligated to the AhCMO gene to construct an intermediate vector pUC 19-AhCMO.
3) The intermediate vector pUC19-AhCMO obtained in step 2) was double digested with EcoRI and HindIII, the expression core fragment containing the AhCMO gene was recovered, and the vector pBI121 after double digestion with EcoRI and HindIII was ligated to each other to construct the overexpression vector pBI121-AhCMO (see FIG. 1).
Example 2AhCMO Gene transformation and identification and screening of transgenic Material
1) AhCMO expression vector for transforming agrobacterium GV3101
And (3) agrobacterium culture: 100uL of prepared agrobacterium GV3101 competent cells are taken, 10ug of pBI121-AhCMO plasmid DNA is added, mixed evenly and placed on ice for 5min to obtain competent mixed liquid. Adding the competence mixed solution into a 2mm electric shock cup (avoiding generating bubbles as much as possible when in use), and electrically shocking at 2500V; quickly adding 800uL YEB liquid culture medium after electric shock, and performing induced culture at 28 deg.C and 180rpm for 5 h; the induced bacterial solution was taken out, spread on YEB plates containing 5Oug/mL each of kanamycin and rifampicin, and cultured at 28 ℃ for 48 hours. Single clones were picked and inoculated into 5mL YEB broth containing kanamycin and rifampicin (both 50ug/mL), shaken at 180rpm for 36 hours, and small amounts of plasmid were extracted for cleavage and PCR identification. The single clones verified to be correct were inoculated into YEB liquid medium containing 5OmL kanamycin and rifampicin at a concentration of 50ug/mL, cultured overnight until OD600 becomes 1.0, added with 50% glycerol, and stored at-80 ℃ until use.
2) Cotton transformation of AhCMO gene
Culturing cotton: seeds of cotton line R15 were provided by the molecular Breeding laboratory of the crop of the institute of Biotechnology, Chinese academy of agricultural sciences, and used with 98% H2SO4Debinding to prepare the starting material. Surface sterilizing the seeds with 70% ethanol for 30s, and immersing in 15% H2O2For 2 hours, followed by 3 rinses with sterile distilled water. Then, the seeds were soaked overnight in sterile water at 28 ℃. After initiation of germination, seed coats were removed, and then the kernels were planted in MS medium and germinated at 28 ℃ under a cycle of 16 hours of light and 8 hours of darkness at a light intensity of 70. mu. mol m-2s-1. Cotyledons and hypocotyls were excised at 5-6 day old seedlings for callus induction explants.
② cotton transformation: the explants are soaked in the agrobacterium suspension for 30 minutes and gently shaken for 3-5 times. Subsequently, infected explants were dried on sterile filter paper and transferred to Callus Induction Medium (CIM) for 2 days, co-cultured at 24 ℃ in the dark. After co-culture, infected explants were transferred to CIM containing 500mg/L cefotaxime and hygromycin at various concentrations and cultured at 28 ℃ under 16 hours light and 8 hours dark cycles for callus induction to obtain 23 transgenic cotton plants.
Positive identification of transgenic cotton: genomic DNA of transgenic cotton plants was extracted for PCR detection. Performing PCR amplification by using AhCMO-F and AhCMO-R as primers,
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’；
PCR reaction system (20 ul): mu.l of template (transgenic cotton genomic DNA), 1. mu.l of each primer, Mix 10. mu.l, sterile water to 20. mu.l.
And (3) PCR reaction conditions: 94 ℃ for 3 min; 94 ℃,30 Sec; 56 ℃,30 Sec; 72 ℃,30 Sec; 30 cycles; 72 ℃ for 10 min.
Results (see FIG. 2) identified 23 transgenic cotton plants, 8 positive plants.
3) qRT-PCR detection of AhCMO gene
RNA was extracted from leaves of transgenic AhCMO lines grown for two weeks at the T2 generation. The extracted RNA is used for reverse transcription, and qRT-PCR is carried out by taking the reverse transcribed cDNA as a template.
A forward primer: 5'-ACCCAACTACTGTTTGTGGAATACC-3'
Reverse primer: 3 '-GTTAACGGGTTTATTAGGAGTACGG-5'
qRT-PCR reaction system (20 ul): mu.l of template (cDNA), 1. mu.l of each primer, KOD-Mix 10. mu.l, and sterile water to 20. mu.l.
qRT-PCR reaction conditions: at 98 ℃ for 2 min; 10Sec at 98 ℃; 30Sec at 60 ℃; 68 ℃ at 30 Sec;
40 cycles; readings were taken every 0.5 ℃ at 65 ℃ to 99 ℃.
The result (shown in figure 3) qRT-PCR detection shows that compared with other 7 positive plants, the expression quantity of AhCMO gene in a transgenic plant is the highest and is named as CMO24 (which is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 2 and 25 months, and the preservation number is CGMCC No. 17381); the expression level of another AhCMO gene is higher, and is second to CMO24, and the gene is named CMO 20.
Example 3AhCMO transgenic plants Low temperature resistance identification test
1) Transgenic cotton CMO24 and wild type cotton R15 were placed under normal growth conditions (28 ℃) for 3 weeks, and as a result (see FIGS. 4-1 and 4-2) leaves of both transgenic and wild type cotton plants appeared normal. The transgenic and wild-type cotton plants were subsequently transferred to 12 ℃ for 24 hours, and as a result, the leaf phenotype of the transgenic cotton CMO24 was found to be normal (see FIGS. 4-4), while the leaves of the non-transgenic plants were severely wilted and even died (see FIGS. 4-3). The fact that the AhCMO transgenic cotton CMO24 is insensitive to low temperature, namely high in low temperature resistance is shown.
2) And (3) placenta blue staining: selecting 3 leaves of wild type R15 and transgenic CMO24 cotton which have similar plant types and sizes and are treated at low temperature, putting the leaves into a 50ml centrifuge tube, and writing numbers. Pouring prepared placenta blue dye solution, vacuum filtering to make the dye solution enter intercellular space, boiling in boiling water for 3-5min, standing at room temperature, and dyeing for 6-8 hr. Decolorizing with 95% ethanol until the tissue is completely transparent. The decolorized leaves were placed on filter paper for observation and photographed. Results prior to cryogenic treatment (see FIGS. 5-1 and 5-2), leaves of wild type R15 and transgenic cotton CMO24 were substantially unstained, while after cryogenic treatment, leaves of non-transgenic cotton R15 were heavily colored (see FIGS. 5-3) and leaves of transgenic cotton CMO24 were relatively lightly colored (see FIGS. 5-4), indicating that cryogenic treatment caused damage to wild type cotton, while cold had less effect on AhCMO transgenic cotton and was highly resistant to cold.
3) Chlorophyll and free nitrogen content determination: before and after the low-temperature treatment, the contents of chlorophyll and free nitrogen in leaves of AhCMO transgenic cotton line CMO24 and wild cotton R15 are measured, and the results show that (see figure 6 and figure 7) before the low-temperature treatment, the contents of chlorophyll and free nitrogen in wild type R15 and transgenic cotton CMO24 are the same; and the chlorophyll and free nitrogen contents of the wild type R15 and the transgenic cotton CMO24 are reduced after the low-temperature treatment, and the reduction amplitude of the transgenic cotton CMO24 is smaller than that of the wild type R15. The result shows that the damage of low-temperature stress on the photosynthesis of cotton is reduced by the expression of the AhCMO gene.
4) And (3) measuring the content of the betaine: betaine content was determined for wild type R15 and AhCMO transgenic cotton CMO24 before and after cryo-treatment, respectively. The determination method comprises the following steps: respectively drying samples of wild type R15 and transgenic cotton CMO24, grinding thoroughly, weighing 0.1g, adding 1ml of extractive solution (80% methanol), extracting at 60 deg.C for 30min, and shaking 1 time every 5 min. Centrifuging at 25 deg.C and 10000rpm for 10min, and collecting supernatant. The measuring tube is 250 mul (supernatant) plus 350 mul (reagent one), the mixture is fully mixed, the mixture reacts for 2 hours at the temperature of 4 ℃, the mixture is centrifuged for 10min at the temperature of 25 ℃ and the rotational speed of 10000rpm, and the supernatant is discarded; adding 300 μ l of 99% diethyl ether, centrifuging at 25 deg.C and 10000rpm for 10min, and placing in a fume hood to volatilize diethyl ether; adding 1ml of 70% acetone, shaking to fully dissolve the precipitate, adjusting the volume of the 1ml cuvette to zero by using the 70% acetone, and measuring the light absorption value at 525 nm.
The results (see fig. 8) showed that after 24 hours of cryogenic treatment, the betaine content in AhCMO transgenic cotton CMO24 was much higher than that of wild-type R15, indicating that overexpression of the AhCMO gene increased the betaine content.
The results show that AhCMO can obviously improve the low temperature resistance of cotton plants.
Example 4 backcross breeding test for Low temperature resistant Cotton varieties Using transgenic Cotton containing AhCMO Gene
The method comprises the following steps:
(1) in 2016, transgenic cotton CMO24 is used as non-recurrent parent and northern Xinjiang early-maturing cotton (self-selecting material in the laboratory) is used as recurrent parent to perform hybridization, and F is obtained1Seed generation;
(2) 2016 planting in winter to Hainan1Second, in the seedling stage, F is extracted1The DNA is used as a substrate to perform PCR amplification by taking AhCMO-F and AhCMO-R as primers; selecting transgenic cotton positive strain (the length of amplified fragment is about 1300bp) to backcross with northern Xinjiang precocious cotton, and harvesting to obtain BC1F1Seed generation; wherein the primer is as follows:
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’；
PCR reaction system (20 ul): mu.l of template (to-be-detected cotton genomic DNA), 1. mu.l of each primer, 10. mu.l of Mix, and 20. mu.l of sterile water. And (3) PCR reaction conditions: 94 ℃ for 3 min; 94 ℃,30 Sec; 56 ℃,30 Sec; 72 ℃,30 Sec; 30 cycles; 72 ℃ for 10 min.
(3) And planting BC in the experiment base of the corridor workshop in the north of Hei in 20171F1Identification of BC by the PCR method described in step (2)1F1Backcrossing the selected positive single plant as male parent with northern Xinjiang early-maturing cotton, and harvesting the single plant to obtain BC2F1Seed generation;
(4) taking single grain as a unit, and carrying out PCR (polymerase chain reaction) on BC according to the step (2)2F1Carrying out PCR identification on the generation seeds, and selecting and reserving positive plants; 2017 in winter, BC is planted in Hainan2F1Backcrossing the selected positive single plant as male parent with northern Xinjiang early-maturing cotton, and harvesting the single plant to obtain BC3F1Seed generation;
(5) taking single grain as a unit, and carrying out PCR (polymerase chain reaction) on BC according to the step (2)3F1Performing PCR identification, selecting positive plants, and planting BC in the test base of the Hebei Gallery in 20183F1Backcrossing the selected single plant as male parent with northern Xinjiang early maturing cotton, and harvesting the single plant to obtain BC4F1Seed generation;
(6) identifying BC by using single grain as unit according to the PCR method in the step (2)4F1Generating plants, selecting positive plants, planting in the south of the Hainan in 2018 winter, selecting plants for selfing, and harvesting according to single plant to obtain BC4F2Seed generation;
(7) taking single seed as unit pair BC4F2And (3) carrying out PCR identification on the generation seeds (the method is shown in step (2)), selecting and reserving corresponding positive strains, and obtaining offspring without isolating AhCMO, namely the bred new variety of the transgenic cotton containing the AhCMO gene, which is named as CMO24 bc.
The CMO24bc is identified at the low temperature of 12 ℃, and the result shows that the CMO24bc has low temperature resistance, which indicates that the backcross breeding method of the invention breeds a new variety with low temperature resistance.
Example 5 Cross-breeding test for Low temperature resistant Cotton varieties Using transgenic Cotton containing AhCMO Gene
The method comprises the following steps:
(1) 2016 in Hebei Gallery test base, transgenic cotton CMO24 was crossed with Su Cotton 12 (laboratory-selected material of the applicant), harvested as F1Seed generation;
(2) 2016 planting in Hainan in winter F1Seed generation, selfing and single plant harvesting to obtain F2Seed generation;
(3) taking single seed as unit, for F2Carrying out PCR identification on generation seeds (see step (2) in example 4), selecting seeds of positive plants (the length of amplified fragments is about 1300bp), planting in the experiment base of the Hebei Gallery in 2017, selecting single plants with good agronomic characters for selfing, and harvesting according to the single plants to obtain F3Seed generation;
(4) repeating the step (3), continuously selecting plants in the Hainan and corridor base for selfing for 2 generations in 2017, except that PCR identification is used for auxiliary selection of AhCMO genes, and the characters of agronomic characters and the like are selected and bred conventionally to obtain F5Generation;
(5) taking single seed as unit, for F5And (3) carrying out PCR identification on the generation seeds, wherein the AhCMO gene in the progeny is not separated, namely the bred new cotton variety containing the AhCMO gene is named as CMO24 f.
(6) The CMO24f is identified at the low temperature of 12 ℃, and the result shows that the low temperature resistance is strong; the new variety containing the AhCMO gene bred by the cross breeding method has low temperature resistance.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences; guoshi pile
<120> breeding method of low-temperature resistant cotton variety
<130> 2019S1471IHCY
<141> 2019-03-12
<160> 2
<170> SIPOSequenceListing 1.0
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atggcagcaa gtgcaacaac aatgttgcta aaatacccaa ctactgtttg tggaatacca 60
aattcatccg caaacaattc tactgatcct tcaaataaca tcgtccaaat tccacaaact 120
actactacta atagcccgct acttaagttc cgtactccta ataaacccgt taacgccgtc 180
gctgccccgg cttttccgtc cgtaaccacc actacaacca ccactccgtc gtccatccaa 240
tcacttgtca aggatttcga tcctcttgtt ccggccgagg atgctcttac tcctcctagc 300
tcttggtata ccgaacctgc cttctatgct catgaacttg accgtatctt ttacaaagga 360
tggcaagtcg cagggtacag tgatcaagtt aaggaggcta accaatattt caccggaacg 420
ttaggaaatg ttgaatattt ggtgtgtcga gatggagaag gaaaagttca tgcatttcac 480
aatgtttgca cccatcgtgc atctatcctt gcttgtggaa gtggcaaaaa gtcatgtttc 540
gtatgccctt atcatggatg ggtatatggc atgaatggat cgcttacgaa agcttcaaaa 600
gcaacaccag aacaatcact aaatcccgat gaacttgggc ttgtaccact aaaagttgca 660
gtatggggcc catttatact catcagtttg gacagatcaa gccgtgaagt aggtgacgtt 720
ggatctgaat ggcttggtag ttgtgctgaa gatgttaagg cccatgcttt tgacccgaat 780
cttcagttca ttaataggag tgaatttcca attgaatcta attggaagat tttcagtgac 840
aactatttgg atagctctta ccatgttcct tatgcacaca aatactatgc aactgagctc 900
gactttgata cttaccaaac cgatatggtt ggaaatgtca cgattcaaag ggtggctggg 960
acttcaaaca atggttttaa tagacttgga actcaagcct tctatgcttt tgcataccct 1020
aactttgctg tggaaaggta tggcccttgg atgactacaa tgcatattgt tccattagga 1080
ccaaggaaat gcaaactagt ggtggactac tatattgaaa aatcaaagct ggacgacaag 1140
gattacatcg aaaagggcat agcaatcaat gataatgtgc agaaagaaga tgtggtgttg 1200
tgtgaaagcg tccaaaaagg gttggagaca ccagcatatc gtagtggaag atatgtgatg 1260
ccaattgaga aaggaattca ccatttccac tgctggttac accaagtgtt gaag 1314
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Ser Leu Val Lys Asp Phe Asp Pro Leu Val Pro Ala Glu Asp Ala Leu
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Gln Val Lys Glu Ala Asn Gln Tyr Phe Thr Gly Thr Leu Gly Asn Val
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Glu Tyr Leu Val Cys Arg Asp Gly Glu Gly Lys Val His Ala Phe His
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Lys Ser Cys Phe Val Cys Pro Tyr His Gly Trp Val Tyr Gly Met Asn
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Phe Ile Leu Ile Ser Leu Asp Arg Ser Ser Arg Glu Val Gly Asp Val
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Claims (2)
1. By using a container containingAhCMOA breeding method for breeding a low-temperature resistant cotton variety by using genetically modified cotton, wherein the breeding method is a backcross breeding method; the backcross breeding method comprises the following steps:
(1) to containAhCMOTransgenic cotton of the gene is used as a non-recurrent parent, a cotton variety with excellent characters is used as a recurrent parent, and F is obtained by hybridization1Generation; said composition containsAhCMOThe transgenic cotton of the gene is CMO24 or contains CMO24AhCMOA derivative line of CMO24 of the gene; accession number of CMO 24: CGMCC No. 17381; the cotton variety with excellent characters refers to popularization and application in productionOr a newly bred cotton line; wherein the characters refer to agronomic characters and fiber quality;
(2) with F1Backcrossing with the cotton variety in step (1) as male parent to obtain BC1F1Generation; taking single seed as unit, extracting BC1F1The single seed genome DNA is substituted and the genome DNA is taken as a substrateAhCMO-F andAhCMOthe R is used as a primer for PCR identification, and offspring with 1314bp PCR amplification product size is selected; backcrossing with the cotton variety of step (1) as female parent to obtain BC2F1Generation; wherein the primer sequence is as follows:
AhCMO-F： 5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R： 3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’；
the reaction system of the PCR comprises: 1 mul of transgenic cotton genome DNA to be detected, 1 mul of each primer, and Mix10 mul are added with sterile water to 20 mul; the reaction conditions of the PCR are as follows: 94 ℃ for 3 min; 94 ℃,30 Sec; 56 ℃,30 Sec; 72 ℃,30 Sec; circulating for 30 times; 72 ℃ for 10 min;
(3) repeating the backcrossing in the step (2) for 3-6 times; finally selfing for 1 generation, performing PCR identification on the seeds of the selfed progeny in the step (2), and reserving the identified single plant progenyAhCMOThe gene is not separated, namely the gene is bred to containAhCMOA novel gene.
2. By using a container containingAhCMOA breeding method for breeding a low-temperature resistant cotton variety by using genetically modified cotton, wherein the breeding method is a cross breeding method; the cross breeding method is characterized by comprising the following steps:
(1) to containAhCMOThe transgenic cotton of the gene is taken as one of the parents, and the excellent cotton variety is taken as the other parent for hybridization to obtain F1Generation; said composition containsAhCMOThe cotton of the gene is CMO 24; accession number of CMO 24: CGMCC No. 17381;
(2) and obtaining F1Selfing and harvesting to obtain F2Generation;
(3) identification and selection of F by molecular marker assistance2Instead withAhCMOPlanting the single grain of the gene to identify the positive descendant, and selecting the plant for selfing; repeating the molecular marker assisted identification and plant selection for selfing for 4-6 generations to obtain the product containingAhCMOA new cotton variety of the gene; wherein the molecular marker-assisted identification is that the genomic DNA of the cotton seed to be detected is taken as a substrate and the genomic DNA is taken as a primerAhCMO-F andAhCMOthe R is used as a primer to carry out PCR amplification, and offspring material with the amplification product size of 1314bp is selected; wherein the primer sequence is as follows:
AhCMO-F：5’-GGCTGCAGGATGGCAGCAAGTGCAACAAC-3’，
AhCMO-R：3’-GGCTCGAGCTTCAACACTTGGTGTAACCAGCAGT-5’。
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| Title |
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| 山菠菜胆碱单加氧酶基因对棉花的遗传转化和耐盐性表达;张慧军 等;《作物学报》;20071231;摘要，摘要以及第1074页右栏 * |
| 罗伯祥 等.转山菠菜胆碱单加氧酶基因（AhCMO）水稻创制及其耐盐性研究.《生命科学研究》.2010,摘要，图1. * |
| 转山菠菜胆碱单加氧酶基因（AhCMO）水稻创制及其耐盐性研究;罗伯祥 等;《生命科学研究》;20100630;摘要，图1 * |
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