CN118291519A - Wx is improvedmpMethod for preparing amylose content of semi-waxy polished round-grained nonglutinous rice - Google Patents
Wx is improvedmpMethod for preparing amylose content of semi-waxy polished round-grained nonglutinous rice Download PDFInfo
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Abstract
The invention provides a method for improving amylose content of Wx mp type semi-waxy polished round-grained nonglutinous rice, which relates to the technical field of bioengineering, and comprises the steps of performing fixed-point editing on two target sequences uORF4 and uORF6 of a Wx mp gene in semi-waxy polished round-grained nonglutinous rice by using a CRISPR/Cas9 gene editing technology, and respectively designing an sgRNA1 target and an sgRNA2 target aiming at the two sequences, wherein the sgRNA1 target sequence is AAAAAATGGATTATATTTCC and is used for knocking out uORF4, and the sgRNA2 target sequence is TAAGTCCTTATAAGCACATA and is used for knocking out uORF6. According to the invention, the CRISPR/Cas9 gene editing technology is used for carrying out fixed-point editing on the specific sequences uORF4 and uORF6 and two sites of the half waxy japonica rice Wx mp gene, so that the amylose content of rice can be improved after the uORF4 and the uORF6 are mutated, and gene resources and technical ideas are provided for improving the rice quality.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for improving the amylose content of Wx mp -type semi-waxy polished round-grained nonglutinous rice.
Background
Rice is a main grain crop in China, and along with the improvement of the living standard of people, the demand is gradually changed from 'full' to 'good', and the consumer has higher and higher requirements on the taste quality of rice. The taste quality of rice is greatly affected by the amylose content of rice, and can be improved by up-regulating or down-regulating the amylose content of rice. Therefore, amylose content has become an important physical and chemical indicator to guide breeders in improving rice varieties.
The amylose content of rice is mainly controlled by Wx, and genotypes of Wx in indica rice, japonica rice and glutinous rice are Wx a、Wxb and Wx respectively. The amylose content of rice in the Wx a background is generally greater than 20%, the amylose content of rice in the Wx b background is generally between 14% and 20%, and the amylose content of rice in the Wx background is generally less than 2%. Generally, the lower the amylose content of rice, the better the taste quality, so breeding workers have long been striving to reduce the amylose content of rice. However, some rice varieties contain a rare Wx genotype, such as Wx mp, whose fourth exon is subjected to a point mutation (G mutation at position 473 is A), resulting in reduced activity of the encoded protein, reduced amylose content (typically less than 10%), and slightly inferior appearance. It is possible to improve the appearance quality by slightly increasing the amylose content in these varieties. Studies show that the translation efficiency of the gene can be improved by editing the uORF region of the gene, so that the gene plays a role in high expression, and the Wx gene is found to contain a plurality of uORF segments. Most of the previous researches focus on editing the coding region and the promoter region of the Wx gene, and editing the target site of the uORF region of the Wx mp gene has not been reported yet.
There have been many studies on the function and application of the rice Wx gene, and the gene has a plurality of natural allelic variation types, including common Wx a,Wxb, wx, unusual Wx mp,Wxmw、Wxlv, etc. However, these types of variation still cannot meet the breeding requirements, and creating new Wx locus variations is important for improving rice quality. The gene editing has great potential in gene research, gene treatment, genetic improvement, etc. because it can perform site-directed genome editing and directional mutation in high efficiency. The traditional rice variety improvement method is long in time consumption and easy to cause change of other characters, and the CRISPR/Cas9 gene editing technology mediated variety improvement and germplasm creation overcome the defects and can realize rapid and precise directional improvement of specific characters. The directional improvement of various properties such as rice quality, fertility, disease resistance and abiotic stress resistance is realized in rice.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for improving the amylose content of the Wx mp semi-waxy polished round-grained nonglutinous rice, which carries out site-directed editing on a specific sequence of a rice Wx gene by using a CRISPR/Cas9 gene editing technology, can increase the amylose content of the rice after gene mutation, and provides gene resources and technical ideas for rice quality improvement research.
The method for improving the amylose content of the Wx mp -type semi-waxy polished round-grained nonglutinous rice comprises an sgRNA1 target and an sgRNA2 target, wherein the sequence of the sgRNA1 target is AAAAAATGGATTATATTTCC, and the sequence of the sgRNA2 target is TAAGTCCTTATAAGCACATA; the sgRNA1 target and the sgRNA2 target are respectively designed by taking two sequences of uORF4 and uORF6 of Wx mp as target sequences, wherein the sequence of the uORF4 is ATGGATTATATTTCCTGGGCTAAAAGAATTGTTGATTTGGCACAATTAAATTCAGTGTCAAGGTTTTGTGCAAGAATTCAGTGTGAAGGAATAGATTCTCTTCAAAACAATTTAATCATTCATCTGATCTGCTCAAAGCTCTGTGCATCTCCGGGTGCAACGGCCAGGATATTTATTGTGCAGTAA,, and the sequence of the uORF6 is ATGGCATTGTAA.
The invention also provides a recombinant vector containing the sgRNA target sequence, and a basic plasmid of the recombinant vector comprises CRISPR/Cas9-U6a-sgRNA1 and CRISPR/Cas9-U6a-sgRNA2.
The invention also provides application of the sgRNA target sequence or the recombinant vector in improving the amylose content of Wx mp -type semi-waxy polished round-grained nonglutinous rice.
The invention also provides application of the sgRNA target sequence or the recombinant vector in editing the Wx mp gene of the half glutinous rice.
The invention also provides application of the sgRNA target sequence or the recombinant vector in editing uORF4 and uORF6 of the Wx mp gene of the half glutinous rice.
Preferably, the editing comprises making deletions and/or insertions and/or substitutions of 1 or more bases in uORF4 and uORF6 of the Wx mp gene of Oryza Glutinosa.
Preferably, the semi-waxy polished round-grained nonglutinous rice is Wx mp type semi-waxy polished round-grained nonglutinous rice.
In the specific creation of the invention, the U6a joint sequence containing the sgRNA target sequence is introduced into receptor rice through an editing vector by constructing a Wx mp gene editing vector to obtain gene editing rice with mutated uORF4 or uORF6 target sites of the Wx mp gene; compared with the acceptor rice, the amylose content of the gene editing rice is obviously improved.
Compared with the prior art, the invention has the beneficial effects that:
1. The CRISPR/Cas9 technology is utilized to successfully edit uORF4 and uORF6 sites of the semi-waxy japonica rice, a novel rice germplasm with obviously improved amylose content is cultivated, and the contribution of the Wx mp gene to the amylose content of rice is proved to have great application value in the field of rice molecular breeding. In the traditional rice amylose directional improvement breeding, hybridization and backcross breeding are mainly adopted, but due to factors such as unstable separation of the progeny thereof, long period, linkage encumbrance of unfavorable genes and the like, it is difficult to cultivate fine varieties capable of stably inheriting and finely adjusting the content of amylose. Therefore, CRISPR/Cas9 technology is widely applied to rice trait improvement and new germplasm creation due to the advantages of simple operation, high editing efficiency, low cost and the like.
2. Different from the prior reports on the aspects of gene editing and target selection, the invention uses the method for editing the uORF4 and the uORF6 sections of the Wx mp genes, not only can ensure the gene editing efficiency, but also can not change the coding region of the Wx mp genes, and can improve the translation efficiency by editing the uORF section of the Wx mp genes, namely, can increase the accumulation of Wx mp proteins, not change the genotype of the coding region, thereby achieving the purpose of finely regulating and controlling the amylose content. Therefore, the CRISPR/Cas9 vector constructed by the invention has important significance and advantages in the aspect of finely improving the amylose of the semi-waxy rice.
Drawings
FIG. 1 is a diagram showing the structure of rice Wx mp gene and the location of sgRNA in the examples of the present invention.
FIG. 2 is a schematic diagram of a single-target PYLCRISPR/Cas9-Wx vector for editing of rice Wx mp gene in an embodiment of the invention.
FIG. 3 is a schematic diagram showing the mutation pattern of Wx mp edited rice in the example of the present invention.
FIG. 4 is a schematic diagram showing amylose content of Wx mp gene-edited rice in the example of the present invention.
Detailed Description
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.
Example 1: target design and gene editing vector construction: the genetic transformation acceptor material selected in the embodiment is a semi-waxy polished round-grained nonglutinous rice variety Nanjing 9108. According to NCBI website prediction, the rice Wx gene is located on the 6 th chromosome of the rice, and the sequence of uORF4 and the sequence of uORF6 of the Wx mp gene of Nanjing 9108 are the same as those of Japanese sunny, and no mutation occurs. And designing sgRNA targets on uORF4 and uORF6 of the Wx gene, wherein each target is respectively provided with a 20bp conserved specific sequence, the nucleotide of the target 1 (sgRNA 1) positioned at the Wx mp gene is positioned at the position from-529 bp to-510 bp, the nucleotide of the target 2 (sgRNA 2) is positioned at the position from-298 bp to-279 bp, and the target position and the sgRNA sequence are shown in figure 1.
According to the requirement of a U6a vector, respectively synthesizing a U6a-sgRNA1-F/R connector and a U6a-sgRNA2-F/R connector, connecting the sgRNA1 with a OsU a plasmid vector and connecting the sgRNA2 with a OsU a plasmid vector after preparing an Oligo dimer, and carrying out two rounds of PCR amplification, wherein primer sequences used in the PCR amplification comprise:
U-F：CTCCGTTTTACCTGTGGAATCG，
sgRNA1(2)-R：CGGAGGAAAATTCCATCCAC，
B1’：TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG，
B2：AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC，
B2’：TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG，
BL：AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC），
The DNA fragments of U6a-sgRNA1 and U6a-sgRNA2 were obtained and then ligated with CRISPR/Cas9 expression vector, specific methods refer to the procedure of (Ma et al, molecular Plant, 2015, 8 (8): 1274-1284), and finally the editing vector PYLCRISPR/Cas9-Wx was successfully constructed, as shown in FIG. 2.
Example 2: identification and editing efficiency analysis of Wx mp gene edited transgenic rice: the agrobacterium EHA105 mediated rice transgenic method is utilized to transform a gene editing vector PYLCRISPR/Cas9-Wx plasmid into a south japonica 9108 callus, and 20 independent T0 transformants are differentiated through hygromycin screening.
The genetic transformation process is as follows: mature embryo callus of japonica rice acceptor variety Nanjing 9108 infected by agrobacterium EHA105 containing PYLCRISPR/Cas9-Wx plasmid is dark-cultured for 3d at 26 ℃ on a co-culture medium, the washed callus is transferred to a selection medium containing hygromycin for resistance screening, the selected resistant callus is transferred to a pre-differentiation medium 14d, then transferred to a differentiation medium for illumination culture, and transferred to a rooting medium for inducing the occurrence of adventitious roots when seedlings grow to 3 cm. When the seedlings grow to 10cm high, the seedlings are taken out, the attached solid culture medium is washed by sterile water and transferred into soil, cultivated in a greenhouse, and after the plants are strong, sampling is carried out for extracting leaf DNA.
PCR detection is carried out by using the primer hygromycin mark (Hyg-F: GCTTTCAGCTTCGATGTAGGAG, hyg-R: CTACACAGCCATCGGTCCAGA) and the nuclease mark (Cas 9-F: GATCCTTACTTTCCGTATTCCTTACTACG, cas9-R: ATACCCTCCTCAATCCTCTTCATG) by taking the south japonica 9108 as a control, and the specific banding agent can be amplified to be a positive transgenic plant. Through identification, 15 positive transgenic plants are screened out, and the positive rate is 75.0%. PCR amplification and sequencing of the target adjacent sequence of the positive transformed plants were performed using primers Wxseq-F/R (Wxseq-F: TTCAACTCTCGTTAAATCATGTCTCT, wxseq-R: GGAGAATTGAAGTTTATTACAATTTGG), and 13 plants were detected to be mutated in the positive transformed plants, with a gene editing efficiency of 86.6%, but no homozygous editing lines were identified in the T 0 transgenic plants, and the editing sites were all heterozygous.
Example 3: screening of Wx mp homozygous editing lines without transgenic components: the gene editing T 0 plant can remove the transgene label hygromycin resistance gene and the nuclease gene through self-exchange in the offspring, thereby obtaining safer editing plant without the transgene label. Thus, to obtain homozygous edited lines without transgenic components, the gene-edited T 0 plants in example 2 were grown up to the T 1 generation.
Leaf DNA of the plants of Nanjing 9108 and T 1 are respectively extracted, molecular identification of transgenic components is carried out by taking Nanjing 9108 as a control and marking with Hyg-F/R and Cas9-F/R, and each plant line is screened out a series of T 1 plants without transgenic components. And screening a homozygous strain with a base deletion through sequencing analysis of a target point adjacent sequence of the plant without the transgenic component, wherein the mutation types of the T 1 target point are consistent with those of the T 0 strain, which shows that the gene editing locus can be inherited stably in different generations.
Example 4: amylose content analysis of gene editing homozygous plants: the grain flour of the Wx mp homozygous edited plant obtained in example 3 was investigated with respect to the south japonica 9108 as a control, and amylose content was measured, and statistical analysis showed that the difference between the amylose content value of the south japonica 9108 and 2 edited plants reached an extremely significant level, and the amplification was 1% -1.5%, as shown in FIG. 4. Therefore, compared with the Nanjing 9108 variety, the Wx mp gene edited rice obtained by the embodiment has obviously improved amylose content and has important value in the field of rice genetic breeding.
The foregoing is only the embodiments of the present invention, and not the limitation of the scope of the present invention, and all equivalent structures made by the content of the present invention are directly or indirectly applied to other related technical fields, which are the same as the scope of the present invention.
Claims (7)
1. The method for improving the amylose content of the Wx mp -type semi-waxy polished round-grained nonglutinous rice is characterized by comprising an sgRNA1 target and an sgRNA2 target, wherein the sequence of the sgRNA1 target is AAAAAATGGATTATATTTCC, and the sequence of the sgRNA2 target is TAAGTCCTTATAAGCACATA; the sgRNA1 target and the sgRNA2 target are respectively designed by taking two sequences of uORF4 and uORF6 of Wx mp as target sequences, wherein the sequence of the uORF4 is ATGGATTATATTTCCTGGGCTAAAAGAATTGTTGATTTGGCACAATTAAATTCAGTGTCAAGGTTTTGTGCAAGAATTCAGTGTGAAGGAATAGATTCTCTTCAAAACAATTTAATCATTCATCTGATCTGCTCAAAGCTCTGTGCATCTCCGGGTGCAACGGCCAGGATATTTATTGTGCAGTAA,, and the sequence of the uORF6 is ATGGCATTGTAA.
2. A recombinant vector comprising the sgRNA target sequence of claim 1, wherein the base plasmid of the recombinant vector comprises CRISPR/Cas9-U6a-sgRNA1, CRISPR/Cas9-U6a-sgRNA2.
3. Use of the sgRNA target sequence of claim 1 or the recombinant vector of claim 2 for increasing the amylose content of Wx mp -type semi-waxy rice.
4. Use of the sgRNA target sequence of claim 1 or the recombinant vector of claim 2 for editing the Wx mp gene of non-glutinous rice.
5. Use of the sgRNA target sequence of claim 1 or the recombinant vector of claim 2 for editing uORF4 and uORF6 of the wu mp gene of japonica rice.
6. The use according to claim 4 or 5, wherein said editing comprises making deletions and/or insertions and/or substitutions of 1 or more bases in uORF4 and uORF6 of the Wx mp gene of non-glutinous rice.
7. The use according to any one of claims 3 to 5, wherein the semi-waxy polished round-grained nonglutinous rice is of the type Wx mp.
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