CN117965566A - Gene family related to tobacco leaf senescence and application thereof - Google Patents
Gene family related to tobacco leaf senescence and application thereof Download PDFInfo
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Abstract
The invention relates to a gene family related to tobacco leaf senescence and application thereof. The gene family includes six genes, nbsgr1.1, nbsgr1.2, nbsgr1.3, nbsgr1.4, nbsgr1.5 and nbsgr1.6, respectively. The biological material developed by utilizing the six genes can carry out polygene editing on tobacco materials, so that six mutants with mutation of all six genes are obtained, and compared with wild type tobacco, the leaf senescence rate of the six mutant tobacco materials is obviously slowed down. The gene family, the protein combination and the related biological materials provided by the invention can be used for creating tobacco materials or tobacco breeding with the effect of delaying leaf senescence.
Description
Technical Field
The invention belongs to the field of tobacco breeding, and particularly relates to a gene family related to tobacco leaf senescence and application thereof.
Background
Plant leaves are important source organs of plants, and provide various nutritional components for the growth and development of plants, including a large amount of energy and organic matters. Whereas senescence in plants was also shown to occur on leaves at the earliest. Leaf senescence is the final stage of plant leaf development, but may lead to premature leaf senescence due to several adverse internal and external factors. Premature leaf senescence causes the crop leaves to prematurely lose photosynthesis and assimilation, shortening leaf functional period, thereby significantly reducing the accumulation of dry matter in the grain, and further resulting in reduced yield and quality. Thus, the leaf senescence process determines to a large extent the yield and quality of crops. And the plant leaf senescence process is delayed, so that the yield can be improved, the nutrient utilization efficiency can be improved, and the method has important significance for agricultural production.
The most remarkable change of leaf senescence is leaf color change, and the principle is that the disintegration of chloroplasts leads to rapid reduction of chlorophyll content, and finally death and shedding of leaves are initiated. The slow-green protein (SGR) is a target protein of chloroplast encoded by nuclear genes, has a highly conserved structure, and is a key factor for chlorophyll degradation and organ senescence in the growth and development process of plants. SGR gene mutations are currently found in a variety of plants resulting in green-retaining mutants such as Arabidopsis thaliana (Arabidopsis thalian), rice (Oryza sativa), alfalfa (Medicagotruncatula), fescue (Festucapratensis) meadow, sorghum (Sorghum bicolor), beans (Phaseolus vulgaris), tomatoes (Solanumlycopersicum), capsicum annuum (Capsicum annuum), soybean (Glycine max) and the like. The molecular mechanism of SGR1 genes in regulating aging is mostly related to chlorophyll degradation or degradation. Mutation of SGR1 gene results in slow degradation rate of chlorophyll in plant, and changes crop yield, quality, biomass and other agronomic characters. In contrast, SGR1 overexpression accelerates the senescence rate of the leaves, and causes the leaves to show yellow and other color changes. The leaf senescence mechanism is damaged by knocking out or mutating key genes in the senescence process through gene editing, so that the senescence of plants can be effectively delayed.
At present, research on molecular mechanisms related to plant leaf senescence regulation has achieved a lot of results, most of the research has focused on identification and functional analysis of leaf senescence-associated genes, and genetic engineering means are utilized to achieve a certain result in delaying plant leaf senescence characteristics. Genes related to leaf senescence reported in tobacco at present are NtCP1, ntCP, ntCP, MC, ntGln1-3, ntGDH1, ntGDH2, ntPSA1, ntH1N1, ntH1N18, ndhF (see literature: gao Xiaoming, guo Yongfeng, china tobacco science 37 (4), tobacco leaf senescence-associated genes), and studies on SGR genes for leaf senescence in tobacco have not been reported at present.
The gene editing technology provides an effective way for solving the senescence of tobacco leaves, but because tobacco is a heterotetraploid plant, the redundancy of gene functions exists among homologous genes, and the ideal effect is difficult to obtain through single gene editing. The multi-gene editing technology can simultaneously interfere a plurality of homologous genes, provides an effective way for solving the inhibition of the homologous genes controlling the same character, and has the problems of non-specificity and incomplete knockout. Therefore, searching all homologous genes for controlling tobacco leaf senescence, and designing target sequence combinations covering all homologous genes, and performing polygene editing on the target sequence combinations through polygene editing technology is an effective method for solving the premature senescence of tobacco leaves.
Disclosure of Invention
In view of the shortcomings of the prior art, it is an object of the present invention to provide a gene family, a protein combination related to tobacco leaf senescence and a biological material related to the gene family and the protein combination.
It is another object of the present invention to provide the use of the gene families, protein combinations and biomaterials described above.
The third object of the present invention is to provide a method for delaying senescence of tobacco leaves using a polygene editing technique.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
The first aspect of the invention provides a gene family related to tobacco leaf senescence, comprising 6 genes; the gene family is designated NbSGR1; the 6 genes are named as NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 respectively; wherein,
The coding sequence (CDS) of the gene nbsgr1.1 is either one of the following (a 1) or (a 2):
(a1) A nucleotide sequence shown as a sequence 1 in a sequence table,
(A2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (a 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence (CDS) of the gene nbsgr1.2 is either (b 1) or (b 2) as follows:
(b1) A nucleotide sequence shown as a sequence 2 in a sequence table,
(B2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (b 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
The coding sequence (CDS) of the gene NbSGR1.3 is any one of the following (c 1) or (c 2):
(c1) A nucleotide sequence shown as a sequence 3 in a sequence table,
(C2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (c 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence (CDS) of the gene NbSGR1.4 is any one of the following (d 1) or (d 2):
(d1) A nucleotide sequence shown as a sequence 4 in a sequence table,
(D2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (d 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence (CDS) of the gene NbSGR1.5 is any one of the following (e 1) or (e 2):
(e1) A nucleotide sequence shown as a sequence 5 in a sequence table,
(E2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (e 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence (CDS) of the gene NbSGR1.6 is any one of the following (f 1) or (f 2):
(f1) A nucleotide sequence shown as a sequence 6 in a sequence table,
(F2) A nucleotide sequence which hybridizes with the nucleotide sequence of (f 1) under stringent conditions and encodes a protein molecule having a function of regulating senescence of tobacco leaves.
The coding sequence of each gene is a nucleotide sequence at the DNA level, and the stringent conditions are as follows: hybridization and washing of membranes with 0.1X SSPE (or 0.1X SSC), 0.1% SDS solution at 65 ℃.
In a second aspect the invention provides a combination of proteins associated with tobacco leaf senescence comprising 6 proteins co-acting to regulate tobacco leaf senescence, the 6 proteins being designated NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 respectively,
The amino acid sequence of the protein NbSGR1.1 is any one of the following (a 3) or (a 4):
(a3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 1 in a sequence table,
(A4) The amino acid sequence shown in the (a 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.2 is any one of the following (b 3) or (b 4):
(b3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 2 in a sequence table,
(B4) The amino acid sequence shown in the (b 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.3 is any one of the following (c 3) or (c 4):
(c3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 3 in a sequence table,
(C4) The amino acid sequence shown in the (c 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
The amino acid sequence of the protein NbSGR1.4 is any one of the following (d 3) or (d 4):
(d3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 4 in a sequence table,
(D4) The amino acid sequence shown in the (d 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
The amino acid sequence of the protein NbSGR1.5 is any one of the following (e 3) or (e 4):
(e3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 5 in the sequence table,
(E4) The amino acid sequence shown in (e 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.6 is any one of the following (f 3) or (f 4):
(f3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 6 in a sequence table,
(F4) The amino acid sequence shown in (f 3) is an amino acid sequence which is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves.
The 6 proteins can be combined to regulate the senescence of tobacco leaves, and when the 6 proteins are mutated simultaneously to cause the loss of functions, the senescence of the tobacco leaves can be delayed.
In a third aspect the present invention provides a biological material associated with the gene family of the first aspect or the protein combination of the second aspect, said biological material being selected from the group consisting of (M) or (N):
(M) a substance for silencing or inhibiting the expression of each gene of the first aspect gene family or each protein of the second aspect protein combination, or a substance for knocking out each gene of the first aspect gene family or each protein of the protein combination of claim 2;
(N) a substance for reducing or inhibiting the activity and/or content of each protein in the combination of proteins of the second aspect.
The biological material can utilize a polygene editing technology, an siRNA technology, an shRNA technology, a transposon technology and the like to mutate related genes of the tobacco material or interfere gene expression, so that the expression of each gene in a gene family is silenced or inhibited, or the activity or content of each protein in a protein combination is reduced or inhibited.
In the biomaterial of the third aspect of the invention, as an alternative, the biomaterial is selected from any one of the following (1) - (4):
(1) A combination of sgRNA target sequences for each gene in the gene family of the first aspect;
(2) The sgRNA combined expression cassette expresses the sgRNA of each gene in the gene family in the first aspect, and the target sequence of the sgRNA is (1) the sgRNA target sequence combination or the reverse complementary sequence of each sequence in the sgRNA target sequence combination;
(3) A vector comprising (2) the sgRNA combination expression cassette;
(4) A host comprising the vector of (3).
The sgRNA combined expression cassette can express a plurality of sgRNAs, and the plurality of sgRNAs can target each gene in the gene family in the first aspect so as to knock out all the genes, thereby achieving the purpose of losing the functions of the genes. In the sgRNA combinatorial expression cassette, 1 or more target sequence binding region-encoding DNA sequences can be designed for each gene, preferably 2 target sequence binding regions-encoding DNA sequences are designed for each gene. The sgRNA combined expression cassette is inserted into a CRISPR/Cas9 vector containing a Cas9 expression cassette, and thus a recombinant expression vector for multi-gene editing can be obtained.
The biological material can also be a transgenic cell line containing the recombinant expression vector, a host bacterium containing the recombinant expression vector and other products which can be used for gene editing.
Further, the sgRNA target sequence combination comprises:
The sgRNA target sequence for the gene nbsgr1.1 includes: the nucleotide sequence of the NbSGR1.1-sgRNA1 is shown as a sequence 7 in a sequence table, and the nucleotide sequence of the NbSGR1.1-sgRNA2 is shown as a sequence 8 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.2 includes: the nucleotide sequence of the NbSGR1.2-sgRNA1 is shown as a sequence 9 in a sequence table, and the nucleotide sequence of the NbSGR1.2-sgRNA2 is shown as a sequence 10 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.3 included: the nucleotide sequence of the NbSGR1.3-sgRNA1 is shown as a sequence 11 in a sequence table, and the nucleotide sequence of the NbSGR1.3-sgRNA2 is shown as a sequence 12 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.4 included: the nucleotide sequence of the NbSGR1.4-sgRNA1 is shown as a sequence 13 in a sequence table, and the nucleotide sequence of the NbSGR1.4-sgRNA2 is shown as a sequence 14 in the sequence table;
the sgRNA target sequence for the gene nbsgr1.5 included: the nucleotide sequence of the NbSGR1.5-sgRNA1 is shown as a sequence 15 in a sequence table, and the nucleotide sequence of the NbSGR1.5-sgRNA2 is shown as a sequence 16 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.6 included: the nucleotide sequence of the NbSGR1.6-sgRNA1 is shown as a sequence 17 in a sequence table, and the nucleotide sequence of the NbSGR1.6-sgRNA2 is shown as a sequence 18 in the sequence table.
Sequences 7 to 18 are nucleotide sequences from the 5 'end to the 3' end.
Further, the vector is designed based on a CRISPR/Cas9 genome editing system, comprising: cas9 expression cassette, above-mentioned sgRNA combination expression cassette, cas9 expression cassette, sgRNA combination expression cassette express a plurality of sgrnas, the target genes of a plurality of said sgrnas are 6 genes in the first aspect gene family.
Further, the vector is pSolycp00032-Cas9-6SGR1-12gRNA, and the nucleotide sequence is shown as sequence 33 in a sequence table (the structure is shown in figure 4).
In a fourth aspect, the present invention provides the use of a gene family according to the first aspect or a protein combination according to the second aspect as defined above for delaying senescence of tobacco leaves, or the use of a biomaterial according to the third aspect for creating a tobacco material or tobacco breeding with a leaf senescence delaying effect.
Knocking out 6 genes in the gene family of the first aspect by using the biological material through a polygene editing technology to lose the functions of the genes, so as to obtain a tobacco material or mutant strain with the effect of delaying leaf senescence; then, the mutant strain is subjected to multi-generation selfing to obtain homozygous mutant strains of 6 genes, and non-transgenic homozygous mutant strains without Cas9 genes can be obtained by continuous identification from the homozygous mutant strains, so that tobacco strains with delayed leaf senescence are obtained.
In a fifth aspect, the present invention provides a method for delaying senescence of tobacco leaves using a polygene editing technique, comprising:
S1: introducing the vector into a tobacco material, and editing 6 genes NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 in the genome of the tobacco material by using a CRISPR/Cas9 genome editing system, so that the functions of the 6 genes are lost;
S2: the transformed plants are obtained through screening, and tobacco mutant strains with obviously delayed leaf senescence are identified from the transgenic plants.
In the method of the fifth aspect of the invention, as an alternative, the method of identifying a tobacco mutant strain having significantly delayed leaf senescence from a transgenic plant comprises: PCR amplification is carried out by taking genome of a transgenic plant as a template, and then detection is carried out by a gel electrophoresis or sequencing method to obtain a strain with mutant genes NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR 1.6;
The primer combination used for PCR amplification comprises the following first primer pair to sixth primer pair:
The first primer pair is: the nucleotide sequence of the NbSGR1.1-sgRNA-F is shown as a sequence 19 in a sequence table, and the nucleotide sequence of the NbSGR1.1-sgRNA-R is shown as a sequence 20 in the sequence table;
the second primer pair is as follows: the nucleotide sequence of the NbSGR1.2-sgRNA-F is shown as a sequence 21 in a sequence table, and the nucleotide sequence of the NbSGR1.2-sgRNA-R is shown as a sequence 22 in the sequence table;
The third primer pair is: the nucleotide sequence of the NbSGR1.3-sgRNA-F is shown as a sequence 23 in a sequence table, and the nucleotide sequence of the NbSGR1.3-sgRNA-R is shown as a sequence 24 in the sequence table;
The fourth primer pair is: the nucleotide sequence of the NbSGR1.4-sgRNA-F is shown as a sequence 25 in a sequence table, and the nucleotide sequence of the NbSGR1.4-sgRNA-R is shown as a sequence 26 in the sequence table;
The fifth primer pair is: the nucleotide sequence of the NbSGR1.5-sgRNA-F is shown as a sequence 27 in a sequence table, and the nucleotide sequence of the NbSGR1.5-sgRNA-R is shown as a sequence 28 in the sequence table;
The sixth primer pair is: the nucleotide sequence of the NbSGR1.6-sgRNA-F is shown as a sequence 29 in a sequence table, and the nucleotide sequence of the NbSGR1.6-sgRNA-R is shown as a sequence 30 in the sequence table.
Sequences 19 to 30 are 5 'to 3' nucleotide sequences.
In a sixth aspect, the invention provides a method of obtaining a non-transgenic tobacco material for delaying senescence of tobacco lamina, comprising:
and (3) carrying out multi-generation selfing on the mutant strain obtained by the method in the fifth aspect, and identifying the selfing progeny which has homozygous mutation of 6 genes in the gene family in the first aspect and does not carry exogenous DNA fragments from the selfing progeny, namely the non-transgenic tobacco material for delaying tobacco leaf senescence.
In a sixth aspect of the invention, the method of authentication further comprises:
A1: taking the genome DNA of the selfing progeny as a template, adopting the first primer pair to the sixth primer pair to perform PCR cloning, electrophoresis and/or sequencing, and selecting a strain in which 6 genes in a gene family are subjected to homozygous mutation, namely, the electrophoresis band is single and different from the electrophoresis band of a wild tobacco plant in size or different from the sequencing result;
A2: and (3) performing PCR amplification and electrophoresis on the Cas9 gene of the homozygous mutant line obtained in the step (A1) by taking the sequence 31 and the sequence 32 in the sequence table as primers, and selecting the homozygous mutant line which does not carry the exogenous Cas9 gene fragment as a non-transgenic tobacco material for delaying tobacco leaf senescence.
Sequences 31 to 32 are nucleotide sequences from 5 'to 3'.
The tobacco in the present invention is preferably a raw tobacco.
Compared with the prior art, the invention has at least the following advantages and beneficial effects:
The invention provides a gene family capable of controlling tobacco leaf senescence, wherein the combination of six genes NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 in the gene family can regulate the tobacco leaf senescence speed. Inhibiting or silencing the expression of the six genes or reducing the content of the protein encoded by the six genes can obviously delay the senescence speed of tobacco leaves.
The present invention also provides a biological material related to the above gene family and protein combination, which can be disabled by rapidly editing six genes, thereby significantly delaying senescence and yellowing of plant leaves. Specifically, the six homologous genes of NbSGR are used as targets, and aging and yellowing of the leaf of the mutant after simultaneous editing of the six homologous genes are delayed.
And thirdly, by utilizing a CRISPR/Cas9 gene editing system, the invention introduces an sgRNA coding sequence containing 12 homologous genes respectively targeting NbSGR1.1-NbSGR1.6 through single transgenosis, thereby rapidly obtaining mutants with all lost functions of six genes, and the nbsgr 1.1.1/2/3/4/5/6 multiple mutant leaves have obvious delay in yellowing and aging.
(IV) six mutants nbsgr 1.1.1/2/3/4/5/6 had significantly delayed leaf senescence relative to the wild type background (Bensheng tobacco), with a chlorophyll content of 26.1 SPAD before dark treatment of the wild type and 17.7 SPAD after 10 days of dark treatment. The chlorophyll content of the six mutants before dark treatment is 26.1 SPAD, and the chlorophyll content after dark treatment for 10 days is 25.3 SPAD.
Drawings
FIG. 1 is a schematic representation of the location of the sgRNA target on the CDS of each gene coding region in example 2 of the present invention.
FIG. 2 is a map of the backbone vector pSolycp00032,00032 plasmid used to construct the polygene editing vectors in example 3 of the present invention.
FIG. 3 is a map of pSolycp00032,00032-Cas 9 plasmid used to construct the multiple gene editing vector in example 3 of the present invention.
FIG. 4 is a schematic representation of the positional structure of the sgRNA combination expression cassette in pSolycp00032-Cas9 plasmid.
FIG. 5 is a map of the final plasmid pSolycp-00032-Cas 9-6NbSGR-12gRNA constructed in example 3 of the present invention.
FIG. 6 is a chart showing phenotype data of six H.benthamiana mutants nbsgr 1.1.1/2/3/4/5/6 obtained in example 4 of the present invention. Wherein A is the wild-type leaf phenotype before dark treatment and B is the wild-type leaf phenotype after 10 days of dark treatment. C is the six mutant (CR 19) leaf phenotype before dark treatment, D is the six mutant (CR 19) leaf phenotype after 10 days of dark treatment.
FIG. 7 is a comparison of chlorophyll content data of six mutants nbsgr 1.1.1/2/3/4/5/6 (CR 19) obtained in example 4 of the present invention and before and after dark treatment of wild type leaves.
FIG. 8 shows the results of examples 4 of the present invention, wherein A is the double leaf phenotype before dark treatment, and B is the double leaf phenotype after 10 days of dark treatment; c is the three-mutant leaf phenotype before dark treatment, D is the three-mutant leaf phenotype after 10 days of dark treatment; e is the four-lobe phenotype before dark treatment, F is the four-lobe phenotype after 10 days of dark treatment; g is the pentalobular phenotype before dark treatment, H is the pentalobular phenotype after 10 days of dark treatment, and the lobular phenotypes before wild-type dark treatment and after 10 days of dark treatment are seen in FIGS. 6A and B.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
The wild type tobacco material used in the following examples is raw tobacco.
Example 1 acquisition of Gene families related to tobacco leaf senescence
(1) On public Bensheng cigarette genome annotation websites (https:// solgenomics. Net /), blast comparison is carried out by taking AtSGR nucleotide sequences as query sequences, and finally 6 genes with high homology with AtSGR1 are obtained, wherein the genes are Niben101Scf02361g03009;Niben101Scf16022g02004;Niben101Scf00490g01040;Niben101Scf09467g02008;Niben101Scf12732g00004;Niben101Scf16022g02005 sequences respectively.
(2) The 6 homologous gene sequences in (1) above were downloaded on a public Bentonia genome annotation website (https:// solgenomics.
Results: the 6 genes were designated NbSGR1.1（Niben101Scf02361g03009）、NbSGR1.2（Niben101Scf16022g02004）、NbSGR1.3（Niben101Scf00490g01040）、NbSGR1.4（Niben101Scf09467g02008）、NbSGR1.5（Niben101Scf12732g00004） and nbsgr1.6 (Niben 101Scf16022g 02005), respectively; wherein CDS of the gene NbSGR1.1 consists of a nucleotide sequence shown as a sequence 1 in a sequence table; CDS of the gene NbSGR1.2 consists of a nucleotide sequence shown as a sequence 2 in a sequence table; CDS of the gene NbSGR1.3 consists of a nucleotide sequence shown as a sequence 3 in a sequence table; CDS of gene NbSGR1.4 is composed of nucleotide sequence shown as sequence 4 in a sequence table; the CDS of the gene NbSGR1.5 consists of a nucleotide sequence shown as a sequence 5 in a sequence table; the CDS of the gene NbSGR1.6 consists of a nucleotide sequence shown as a sequence 6 in a sequence table.
EXAMPLE 2 sgRNA target sequence design of the genes NbSGR1.1-NbSGR1.6
The method comprises the following steps of:
For the 6 homologous genes obtained in the example 1, the website CRISPR-GE (http:// skl. Scau. Edu. Cn /) is used for designing the sgRNA, 2 sgRNA targets are designed for each gene, 12 sgRNA targets are used for selecting the sgRNA targets according to the targeting efficiency and the off-targeting condition and comprehensively considering the conditions of six targeting genes, the positions of the sgRNA targets of the genes on the coding regions of the corresponding genes are shown in figure 1, the sgRNA target sequences of the genes are as follows, and the corresponding gene names contained in the target sequence names indicate that the target sequences are designed for the genes.
NbSGR1.1-sgRNA1:5'-AAAGAGAGCTTTCCTTATGA-3' (sequence 7 in the sequence Listing);
NbSGR1.1-sgRNA2:5'-TTAGATGCTTCAAAAATAGA-3' (sequence 8 in the sequence Listing);
NbSGR1.2-sgRNA1:5'-AACTATGGACTGATTCTTGT-3' (sequence 9 in the sequence Listing);
NbSGR1.2-sgRNA2:5'-TGTTACCTCTAAACTCACTT-3' (sequence 10 in the sequence Listing);
NbSGR1.3-sgRNA1: 5'-GCGGAAAATTACAGGGACAA-3' (sequence 11 in the sequence Listing);
NbSGR1.3-sgRNA2:5'-TTTGATGCTTCAAATATAGC-3' (sequence 12 in the sequence Listing);
NbSGR1.4-sgRNA1:5'-CATACTCCAGTATCGTGCCA-3' (sequence 13 in the sequence Listing);
NbSGR1.4-sgRNA2:5'-AATGTCATACCTGATCGTAT-3' (SEQ ID NO:14 in the sequence Listing);
NbSGR1.5-sgRNA1:5'-AAGACAAGGGTGTTGTAAGG-3' (sequence 15 in the sequence Listing);
NbSGR1.5-sgRNA2:5'-TATTGAAGCTGGATCAAGAT-3' (sequence 16 in the sequence Listing);
NbSGR1.6-sgRNA1:5'-TAGACTGCACAGAGATGAAG-3' (SEQ ID NO:17 in the sequence Listing);
NbSGR1.6-sgRNA2:5'-GCAGAAGATATAACAAGAAA-3' (SEQ ID NO:18 of the sequence Listing).
In addition, 3' connected PAM sequences of target sequences NbSGR1.1-sgRNA1, nbSGR1.5-sgRNA1 and NbSGR1.5-sgRNA2 are GGG; the 3' connected PAM sequence of the target sequence NbSGR1.1-sgRNA2 is AGG; target sequences NbSGR1.2-sgRNA1, nbSGR1.2-sgRNA2, nbSGR1.3-sgRNA1, nbSGR1.3-sgRNA2, nbSGR1.4-sgRNA2, nbSGR1.6-sgRNA1 and 3' connected PAM sequences of NbSGR1.6-sgRNA2 are TGGs; the 3' linked PAM sequence of NbSGR1.4-sgRNA1 is CGG.
If the PAM sequence is located at the 5' -end, the PAM sequence is the reverse complement sequence of the corresponding PAM sequence.
Example 3 NbSGR1.1-NbSGR1.6 Multi-Gene editing vector pSolycp00032-Cas9-6NbSGR-
Construction of 12gRNA
The method is carried out according to the following steps:
(1) Nucleic acid fragments containing 12 sgRNA target sequences or coding DNA sequences containing sgrnas that bind to 12 target sequences were designed. The nucleic acid fragment is designed as follows: for each target site, two complementary DNA sequences are designed, a homologous arm F sequence, a tRNA sequence and a gRNA scaffold sequence are respectively added at the 5 'end and the 3' end of the target sequence, the homologous arm R sequence is underlined to form 12 sgRNA target sequences provided in example 2, the specific design principle is referred to in literature (KabinXie, Bastian Minkenberg, and Yinong Yang,P Natl Acad Sci USA,112, 3570-3575.Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system)., the nucleic acid fragment is artificially synthesized, and the sequence of the synthesized nucleic acid fragment is shown as follows (namely the sequence in a sequence table 34）：5'-ggccagtgccaagcttcgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagtttttttttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgattgAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAAAGAGAGCTTTCCTTATGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATTAGATGCTTCAAAAATAGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAACTATGGACTGATTCTTGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATGTTACCTCTAAACTCACTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGCGGAAAATTACAGGGACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATTTGATGCTTCAAATATAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACATACTCCAGTATCGTGCCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAATGTCATACCTGATCGTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAAGACAAGGGTGTTGTAAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATATTGAAGCTGGATCAAGATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCATAGACTGCACAGAGATGAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGCAGAAGATATAACAAGAAAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttttgcaaaattttccagatcgatttcttcttcctctgttcttcggcgttcaatttctggggttttctcttcgttttctgtaactgaaacctaaaatttgacctaaaaaaaatctcaaataatatgattcagtggttttgtacttttcagttagttgagttttgcagttccgatgagataaaccaataagcttgcatgcctgc-3'
(2) The CRISPR/Cas9 vector system is adopted, and comprises a vector pSolycp00032-Cas9, wherein the plasmid map of the vector is shown in figure 3, a skeleton vector pSolycp00032 of the vector can be purchased from Newton's biological company (product number: V003093 #), the plasmid map of the vector is shown in figure 2, the modified pSolycp00032-Cas9 (namely 23kn175 in figure 3) is stored in a cotton biotechnology breeding innovation research group of China agricultural sciences, and the public can obtain the vector from the laboratory. FIG. 4 is a schematic diagram of the positional structure of the sgRNA combinatorial expression cassette. The sgRNA combination expression cassette was inserted into vector pSolycp00032-Cas9 at the Hind iii cleavage site, thereby obtaining the multi-gene editing vector of the invention. The method comprises the following steps:
The backbone vector pSolycp-00032 was digested with PstI and EcoRI, and then the Cas9 expression cassette backbone vector fragment was inserted into the digested backbone vector through PstI and EcoRI cleavage sites, thereby obtaining pSolycp-00032-Cas 9. The nucleotide sequence of the Cas9 expression cassette is shown as a sequence 33 in a sequence table. Vector pSolycp00032-Cas9 was digested with NEB company Hind iii (see NEB functional net for digestion conditions) to form a large fragment of vector with cohesive ends;
PCR amplification (2-step amplification method in the specification of the amplification conditions reference high-fidelity enzyme KOD-Plus-Neo) was performed using the synthesized nucleic acid fragment of the above (1) as a template and 12gRNA-F and 12gRNA-R as primers with the high-fidelity enzyme KOD-Plus-Neo (product number: KOD-401) of TOYOBO company, to obtain an 2724bp amplification product;
12 gRNA-F5'-ggccagtgccaagcttcgacttgccttcc-3' (sequence 35 in the sequence Listing);
12 gRNA-R5'-gcaggcatgcaagcttattggtttatctcatcgg-3' (SEQ ID NO: 36 in the sequence Listing).
Then, the large fragment of the vector after cleavage and the amplified product were mixed at a molar concentration of about 1:5, and subjected to ligation using the In-Fusion HD Cloning Kit (cat# 639648) from TaKaRa company (ligation procedure is referred to In the specification), E.coli DH 5. Alpha. Was transformed to obtain a ligated plasmid, which was sequenced using the common primer M13, and the correct plasmid clone was selected. Finally, the target recombinant plasmid, namely the polygene editing vector pSolycp00032-Cas9-6NbSGR-12gRNA, is obtained (figure 5).
EXAMPLE 4 obtaining of polygene editing transgenic plants
1. Transforming recombinant plasmid into agrobacterium
1. Mu.g of the recombinant plasmid pSolycp-00032-Cas 9-6NbSGR-12gRNA prepared in example 3 was placed in 100. Mu.L of GV3101 competent cells (Beijing Wash Vietnam, NRR 01270), quick-frozen in liquid nitrogen for 3 minutes, water-bath at 37℃for 5 minutes, then 1mL YEB medium (YEB medium consisting of solute and solvent, the solvent being water, the concentration of solute and concentration in the medium being 5g/L of yeast extract, 5g/L of peptone, 5g/L of beef extract, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of sucrose) was added, and incubated at 28℃for 2-4 hours; centrifugation at 10000 Xg for 30 seconds, discarding the supernatant, adding 0.1mL of YEB medium to re-suspend the cells, plating on YEB plates containing 50. Mu.g/mL kanamycin, 500. Mu.g/mL streptomycin, and 50. Mu.g/mL rifampicin, and culturing in the dark at 28℃for 2-3 days; selecting single colonies, inoculating the single colonies into a YEB liquid culture medium containing 50 mug/mL kanamycin, 500 mug/mL streptomycin and 50 mug/mL rifampicin, and carrying out shake culture at 28 ℃ overnight to obtain transformants; and (3) carrying out bacterial liquid PCR identification on the transformant, taking hpt557-F and hpt557-R as primers, obtaining an amplified fragment with the size of 557bp, namely positive recombinant bacteria, and freezing at-80 ℃ for later use.
Hpt557-F: 5'-ACACTACATGGCGTGATTTCAT-3' (sequence 31 in the sequence Listing);
hpt557-R: 5'-TCCACTATCGGCGAGTACTTCT-3' (SEQ ID NO: 32 in the sequence Listing).
2. Obtaining of T0 generation regenerated plants
(1) The leaf of the benthonic tobacco planted in the culture medium with the age of 4-6 weeks is used as the infection material. Agrobacterium GV3101 with pSolycp00032-Cas9-6NbSGR-12g RNA plasmid was removed from the-80℃refrigerator, streaked activated and then selected for monoclonal culture in 100ml LB medium containing the corresponding antibiotic (100 ug/mlKan,50 ug/mlRif), 28℃overnight. 5000Xg and 5min were collected. The cells were resuspended to approximately od600=0.6 with MS medium.
(2) Cutting the sterile tobacco leaf into 0.5cm by 0.5cm, soaking in the re-suspended bacterial liquid in step (1) for 10min, and shaking for several times. The leaf was removed, the leaf surface bacteria solution was blotted with filter paper, and the leaf was plated on a co-culture medium (containing 4.4g MS powder, 30g sucrose, 0.5mg IAA,2mg 6-BA per liter) and dark-cultured for 2 days.
(3) The dark cultured leaves were transferred to a differentiation medium (4.4 g MS powder per liter, 30g sucrose, 500mgcef,100mg Kan,0.5mg IAA,2mg 6-BA) and the differentiation medium was changed every 10 days during the culture until the shoots were differentiated.
(4) When the buds grow to about 2cm, cutting off the buds, transferring the buds to a rooting culture medium (containing 4.4g MS powder and 30g sucrose per liter, 250mg cef,100mg Kan,0.5mg IAA), discarding the buds which are not rooted yet for 20 days, transferring the rooted seedlings, namely potential positive seedlings, into soil for planting, and sampling for detection after the seedling state is stable.
3. Molecular detection of transgenic seedlings
Extracting genome DNA from the obtained T0 potential positive seedlings and wild type benthamia tabacum, directly designing primers at two sides of each target site, performing PCR amplification by Genstar company 2X Taq PCR StarMix (product number: A012) (amplification conditions refer to 2X Taq PCR StarMix specification), performing a PCR system by one pair of primers, sequencing the corresponding amplified products obtained after amplification of each pair of primers, and detecting the primer sequences as follows (sequences 19-30 in a sequence table):
NbSGR 1.1-sgRNA-F5'-AGGAAAAGCGAGTACGCAAG-3' (sequence 19 in the sequence Listing)
NbSGR 1.1-sgRNA-R5'-CTGTAGACCCTCCGCTCAAG-3' (SEQ ID NO: 20)
NbSGR 1.2-sgRNA-F5'-AGGAAATCCTTTGCCCACTT-3' (sequence 21 in the sequence Listing)
NbSGR 1.2-sgRNA-R5'-ATTAGAGAGCCAGCGCGATA-3' (sequence 22 in the sequence Listing)
NbSGR 1.3-sgRNA-F5'-TGAAATCGGAACTTTGACTGC-3' (sequence 23 in the sequence Listing)
NbSGR 1.3-sgRNA-R5'-ACGGGTCCGACACCTATTTT-3' (sequence 24 in the sequence Listing)
NbSGR 1.4-sgRNA-F5'-TGCCTACTAAGACGATCATTTCTG-3' (SEQ ID NO: 25)
NbSGR 1.4-sgRNA-R5'-TCGAGGGCTTTGGTCTCTAA-3' (SEQ ID NO: 26)
NbSGR 1.5-sgRNA-F5'-CCGCATCCACATATTCTCCT-3' (sequence 27 in the sequence Listing)
NbSGR 1.5-sgRNA-R5'-TGCCGTAAAGGAAATTCAGG-3' (SEQ ID NO: 28)
NbSGR 1.6-sgRNA-F5'-GCAGTTAAAAGGATGGCACC-3' (SEQ ID NO: 29)
NbSGR 1.6-sgRNA-R5'-GGGGCCCATGTATCTCACTA-3' (SEQ ID NO: 30 in the sequence Listing).
A pair of primers, a PCR system, and the primer pair is used for carrying out PCR by taking a wild-type benthonic tobacco genome as a template, and the obtained amplified product has the following sequence:
The sequence of the PCR product of the primer pair consisting of the sequence 19 and the sequence 20 is shown as a sequence 37 in a sequence table;
the sequence of the PCR product of the primer pair consisting of the sequence 21 and the sequence 22 is shown as a sequence 38 in a sequence table;
the sequence of the PCR product of the primer pair consisting of the sequence 23 and the sequence 24 is shown as a sequence 39 in a sequence table;
the sequence of the PCR product of the primer pair consisting of the sequence 25 and the sequence 26 is shown as a sequence 40 in a sequence table;
the sequence of the PCR product of the primer pair consisting of the sequence 27 and the sequence 28 is shown as a sequence 41 in a sequence table;
The sequence of the PCR product of the primer pair consisting of the sequence 29 and the sequence 30 is shown as a sequence 42 in a sequence table;
Results: comparing the sequencing sequence of the PCR product obtained by each primer pair in the transgenic seedling with the sequencing sequence of the PCR product of the corresponding primer pair of wild type tobacco, analyzing mutation conditions, and finding that mutation can be detected at all 12 sgRNAs, wherein the mutation types are mostly in the form of complex mutation (Complicated Variant, CV), single base or multiple base insertion (in) or deletion (del). CR19 in the T0 transgenic Line is a mutant (such as Line16 in the following) with mutations in all six genes, also called a six mutant, which significantly delayed the rate of leaf senescence yellowing under dark treatment compared to the wild type, see in particular FIGS. 6 and 7.
In addition to the six mutants, the test also obtained 5 strains (two mutants (Line 28), three mutants (Line 24), four mutants (lines 12 and 18) and five mutants (Line 19)) of the T0-generation smoke mutant, the detection results of the mutation at two target sites of each gene of each mutant strain are shown in Table 1, specifically, the detection results are compared with the sequencing results of the PCR products of the WT according to the sequencing results of the PCR products of the respective strains, the detection results are expressed by WT when no mutation occurs at all target sites, the detection results are expressed by in when single base or multiple base insertion occurs at the target sites, the detection results are expressed by del when single base or multiple base deletion occurs at the target sites, the detection results are expressed by CV when complex mutation occurs at the target sites, and the complex mutation is expressed by complex mutation except single base or multiple base insertion (in) or deletion (del). The mutation at two target sites for each gene is given in table 1.
4. Acquisition of homozygous mutant Using transgenic Positive mutant
The T0 generation transgenic strain CR19 is self-pollinated to reproduce two generations, each generation uses the NbSGR 1.1-NbSGR 1.6 detection primer (sequence 19-30) to detect mutation condition, and simultaneously uses primer pairs hpt557-F (sequence 31) and hpt557-R (sequence 32) to detect whether the transgenic vector is still in genome or not, if PCR product does not contain 557bp fragment, the transgenic vector is not in genome, the plant is proved to be a non-transgenic plant, if the transgenic vector is proved to be not in plant and each gene of the plant is homozygous mutation, the non-transgenic homozygous mutant is obtained.
Example 5 phenotypic observations and chlorophyll content determination tests of leaves of transgenic plants obtained after polygene editing
The test method comprises the following steps:
Darkness is an effective external stimulus for promoting leaf senescence, and therefore, dark culture is often applied to senescence research as an effective means for leaf senescence simulation. Planting the T0 generation mutant materials in the table 1 in a greenhouse, taking leaves with the same development age at the same part for dark treatment in the 6-leaf period, and holding the leaf stalks by wet cotton balls to avoid the leaf wilting due to water loss. Leaf chlorophyll content was measured with SPAD-502 before and after treatment, respectively.
Results: after 10 days of dark culture, wild-type leaves significantly yellow senescent (FIG. 6A, B), nbSGR six mutant CR19 leaves remained green (FIG. 6C, D). The chlorophyll content measurement results are consistent with the phenotype, and as shown in fig. 7, the chlorophyll content reduction rate of the CR19 leaf is remarkably slowed down. Other double (FIG. 8A, B), triple (FIG. 8C, D), tetradentate (FIG. 8E, F), pentadentate (FIG. 8G, H) mutants such as those of lines 28, 24, 18 and 19 also turned yellow after 10 days of dark culture, which were not substantially different from wild-type leaves, see FIG. 8 in particular. It is shown that six mutants obtained by editing these 6 genes by the method of the present invention have the characteristic of significantly delaying leaf senescence.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A family of genes associated with tobacco leaf senescence, said family of genes comprising 6 genes; the gene family is designated NbSGR1; the 6 genes are named as NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 respectively; wherein,
The coding sequence of the gene NbSGR1.1 is any one of the following (a 1) or (a 2):
(a1) A nucleotide sequence shown as a sequence 1 in a sequence table,
(A2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (a 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
The coding sequence of the gene NbSGR1.2 is any one of the following (b 1) or (b 2):
(b1) A nucleotide sequence shown as a sequence 2 in a sequence table,
(B2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (b 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
The coding sequence of the gene NbSGR1.3 is any one of the following (c 1) or (c 2):
(c1) A nucleotide sequence shown as a sequence 3 in a sequence table,
(C2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (c 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence of the gene NbSGR1.4 is any one of the following (d 1) or (d 2):
(d1) A nucleotide sequence shown as a sequence 4 in a sequence table,
(D2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (d 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence of the gene NbSGR1.5 is any one of the following (e 1) or (e 2):
(e1) A nucleotide sequence shown as a sequence 5 in a sequence table,
(E2) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence of (e 1) and encodes a protein molecule having a function of regulating senescence of tobacco leaves;
the coding sequence of the gene NbSGR1.6 is any one of the following (f 1) or (f 2):
(f1) A nucleotide sequence shown as a sequence 6 in a sequence table,
(F2) A nucleotide sequence which hybridizes with the nucleotide sequence of (f 1) under stringent conditions and encodes a protein molecule having a function of regulating senescence of tobacco leaves.
2. A combination of proteins associated with tobacco leaf senescence comprising 6 proteins which co-act to regulate tobacco leaf senescence, the 6 proteins being designated NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR1.6 respectively, wherein,
The amino acid sequence of the protein NbSGR1.1 is any one of the following (a 3) or (a 4):
(a3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 1 in a sequence table,
(A4) The amino acid sequence shown in the (a 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.2 is any one of the following (b 3) or (b 4):
(b3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 2 in a sequence table,
(B4) The amino acid sequence shown in the (b 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.3 is any one of the following (c 3) or (c 4):
(c3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 3 in a sequence table,
(C4) The amino acid sequence shown in the (c 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
The amino acid sequence of the protein NbSGR1.4 is any one of the following (d 3) or (d 4):
(d3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 4 in a sequence table,
(D4) The amino acid sequence shown in the (d 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
The amino acid sequence of the protein NbSGR1.5 is any one of the following (e 3) or (e 4):
(e3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 5 in the sequence table,
(E4) The amino acid sequence shown in (e 3) is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves;
the amino acid sequence of the protein NbSGR1.6 is any one of the following (f 3) or (f 4):
(f3) An amino acid sequence encoded by a nucleotide sequence shown as a sequence 6 in a sequence table,
(F4) The amino acid sequence shown in (f 3) is an amino acid sequence which is subjected to substitution, deletion or addition of one or more amino acid residues and has the function of regulating and controlling the senescence of tobacco leaves.
3. A biological material associated with the gene family of claim 1 or the protein combination of claim 2, said biological material being selected from the group consisting of (M) or (N):
(M) a substance for silencing or inhibiting the expression of the individual genes of the gene family of claim 1 or the individual proteins of the protein combination of claim 2, or a substance for knocking out the individual genes of the gene family of claim 1 or the individual proteins of the protein combination of claim 2;
(N) a substance for reducing or inhibiting the activity and/or content of the individual proteins in the protein combination according to claim 2.
4. A biomaterial according to claim 3, wherein the biomaterial is selected from any one of the following (1) - (4):
(1) A combination of sgRNA target sequences for each gene in the gene family of claim 1;
(2) An sgRNA combinatorial expression cassette that expresses a target of the sgRNA of each gene in the gene family of claim 1, the target sequence of the sgRNA being (1) the sgRNA target sequence combination or the reverse complement of each sequence in the sgRNA target sequence combination;
(3) A vector comprising (2) the sgRNA combination expression cassette;
(4) A host comprising the vector of (3).
5. The biomaterial of claim 4, wherein the sgRNA target sequence combination comprises:
The sgRNA target sequence for the gene nbsgr1.1 of claim 1 comprising: the nucleotide sequence of the NbSGR1.1-sgRNA1 is shown as a sequence 7 in a sequence table, and the nucleotide sequence of the NbSGR1.1-sgRNA2 is shown as a sequence 8 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.2 of claim 1 comprising: the nucleotide sequence of the NbSGR1.2-sgRNA1 is shown as a sequence 9 in a sequence table, and the nucleotide sequence of the NbSGR1.2-sgRNA2 is shown as a sequence 10 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.3 of claim 1 comprising: the nucleotide sequence of the NbSGR1.3-sgRNA1 is shown as a sequence 11 in a sequence table, and the nucleotide sequence of the NbSGR1.3-sgRNA2 is shown as a sequence 12 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.4 of claim 1 comprising: the nucleotide sequence of the NbSGR1.4-sgRNA1 is shown as a sequence 13 in a sequence table, and the nucleotide sequence of the NbSGR1.4-sgRNA2 is shown as a sequence 14 in the sequence table;
The sgRNA target sequence for the gene nbsgr1.5 of claim 1 comprising: the nucleotide sequence of the NbSGR1.5-sgRNA1 is shown as a sequence 15 in a sequence table, and the nucleotide sequence of the NbSGR1.5-sgRNA2 is shown as a sequence 16 in the sequence table;
the sgRNA target sequence for the gene nbsgr1.6 of claim 1 comprising: the nucleotide sequence of the NbSGR1.6-sgRNA1 is shown as a sequence 17 in a sequence table, and the nucleotide sequence of the NbSGR1.6-sgRNA2 is shown as a sequence 18 in the sequence table.
6. The biomaterial of claim 4 or 5, wherein the vector is designed based on a CRISPR/Cas9 genome editing system, comprising: a Cas9 expression cassette, the sgRNA combination expression cassette of claim 4, the Cas9 expression cassette expressing Cas9, the sgRNA combination expression cassette expressing a plurality of sgrnas, the target genes of the plurality of sgrnas being 6 genes in the gene family of claim 1.
7. Use of the gene family of claim 1 or the protein combination of claim 2 for delaying senescence of tobacco leaves, or use of the biomaterial of any of claims 3-6 for creating a tobacco material or tobacco breeding with a leaf senescence delaying effect.
8. A method for delaying senescence of tobacco leaves using a polygene editing technique, the method comprising:
S1: introducing the vector of claim 6 into a tobacco material, editing the 6 genes nbsgr1.1, nbsgr1.2, nbsgr1.3, nbsgr1.4, nbsgr1.5 and nbsgr1.6 in the genome of the tobacco material using a CRISPR/Cas9 genome editing system, thereby losing the 6 genes;
S2: the transformed plants are obtained through screening, and tobacco mutant strains with obviously delayed leaf senescence are identified from the transgenic plants.
9. The method of claim 8, wherein the method of identifying a tobacco mutant having significantly delayed leaf senescence from a transgenic plant comprises: PCR amplification is carried out by taking genome of a transgenic plant as a template, and then detection is carried out by a gel electrophoresis or sequencing method to obtain a strain with mutant genes NbSGR1.1, nbSGR1.2, nbSGR1.3, nbSGR1.4, nbSGR1.5 and NbSGR 1.6;
The primer combination used for PCR amplification comprises the following first primer pair to sixth primer pair:
The first primer pair is: the nucleotide sequence of the NbSGR1.1-sgRNA-F is shown as a sequence 19 in a sequence table, and the nucleotide sequence of the NbSGR1.1-sgRNA-R is shown as a sequence 20 in the sequence table;
the second primer pair is as follows: the nucleotide sequence of the NbSGR1.2-sgRNA-F is shown as a sequence 21 in a sequence table, and the nucleotide sequence of the NbSGR1.2-sgRNA-R is shown as a sequence 22 in the sequence table;
The third primer pair is: the nucleotide sequence of the NbSGR1.3-sgRNA-F is shown as a sequence 23 in a sequence table, and the nucleotide sequence of the NbSGR1.3-sgRNA-R is shown as a sequence 24 in the sequence table;
The fourth primer pair is: the nucleotide sequence of the NbSGR1.4-sgRNA-F is shown as a sequence 25 in a sequence table, and the nucleotide sequence of the NbSGR1.4-sgRNA-R is shown as a sequence 26 in the sequence table;
The fifth primer pair is: the nucleotide sequence of the NbSGR1.5-sgRNA-F is shown as a sequence 27 in a sequence table, and the nucleotide sequence of the NbSGR1.5-sgRNA-R is shown as a sequence 28 in the sequence table;
The sixth primer pair is: the nucleotide sequence of the NbSGR1.6-sgRNA-F is shown as a sequence 29 in a sequence table, and the nucleotide sequence of the NbSGR1.6-sgRNA-R is shown as a sequence 30 in the sequence table.
10. The use according to claim 7 or the method according to claim 8 or 9, wherein the tobacco is a raw tobacco.
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