CN114990153A

CN114990153A - Application of rice lipid transfer protein in improving fatty acid content and reducing chalkiness of rice

Info

Publication number: CN114990153A
Application number: CN202210545399.6A
Authority: CN
Inventors: 肖浪涛; 刘清; 申鹏辉; 谢琴; 王若仲; 黄志刚
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2022-09-02
Anticipated expiration: 2042-05-19
Also published as: CN114990153B

Abstract

The invention discloses application of rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness degree of rice. The rice mutant with OsLTP10 protein or OsLTP24 knocked out shows that the content of fatty acid is reduced, the chalkiness of grains is increased, and the rice quality is deteriorated; and the chalkiness of over-expression strain seeds is obviously reduced, the content of fatty acid is increased, and the rice quality is improved, which shows that the OsLTP10 protein and the OsLTP24 protein have important functions for improving the rice quality. The invention provides a new thought for the research on the molecular mechanism of chalkiness and fatty acid content of rice and also provides a new gene resource for improving the quality of rice.

Description

Application of rice lipid transfer protein in improving fatty acid content of rice and reducing chalkiness of rice

Technical Field

The invention belongs to the technical field of rice breeding, and particularly relates to application of rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness degree of the rice.

Background

With the improvement of living standard, people have higher and higher requirements on the quality of rice. Southern early indica rice has excellent properties of high yield, strong resistance and the like, but the quality of the early indica rice is relatively poor, so that the planting economic benefit is low.

The rice nutrient components mainly comprise starch, protein, fatty acid and the like. Although lipids are abundant as a 3 rd nutrient in rice, the fat content of rice is low. The content and components of unsaturated fatty acid or starch-fat complex in rice can affect the quality of rice, and the components have high nutritive value for human body and good prevention effect on cardiovascular diseases. The main components of the fatty acid of rice are oleic acid, linoleic acid and palmitic acid, and account for about 90 percent of the total fatty acid content. In general, rice with higher fat content has better taste quality. Within a certain range, the rice with high fat content has better integrity, higher glossiness, palatability and aroma. However, the content of crude fat in rice is low, extraction is difficult, previous researches on rice fat mostly focus on the relationship between the rice fat and quality and the fatty acid composition of the rice, and the researches on the content of the rice fat and the regulation and control of the composition are less. Meanwhile, the chalkiness proportion and the whole rice rate of the rice are two key factors for determining the market price of the rice, and the chalkiness indirectly influences the whole rice rate.

Therefore, the reduction of chalkiness and the improvement of the fatty acid content of rice are the main directions of rice quality breeding, so that the quality gene function research is carried out, the new variety breeding and the rice industrialization of the high-quality rice of the rice are promoted, and the grain production benefit is further improved.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness degree of the rice, and the invention can effectively improve the fatty acid content of the rice and reduce the chalkiness degree of the rice.

The invention also provides application of the nucleic acid molecule for encoding the rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness of the rice.

The invention also provides biological materials related to the nucleic acid molecules.

The invention also provides a primer set for amplifying a nucleic acid molecule encoding OsLTP10 protein or a nucleic acid molecule encoding OsLTP24 protein.

The invention also provides a method for cultivating the rice with high fatty acid content.

The invention also provides a method for reducing the chalkiness degree of rice.

In a first aspect of the invention, the use of a rice lipid transfer protein, OsLTP10 protein and OsLTP24 protein, for increasing the fatty acid content and reducing the chalkiness of rice is proposed.

In some embodiments of the invention, the OsLTP10 protein is a protein of a) or b) or c) or d) as follows:

a) protein with amino acid sequence shown as SEQ ID NO. 2;

b) the fusion protein is obtained by connecting a label at the N end and/or the C end of the protein with the amino acid sequence shown as SEQ ID NO. 2;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown as SEQ ID NO. 2;

d) protein with 75% or more than 75% homology with amino acid sequence shown as SEQ ID NO. 2 and with the same function.

In some embodiments of the invention, the OsLTP24 protein is a protein of e) or f) or h) or i) as follows:

e) protein with amino acid sequence shown as SEQ ID NO. 4;

f) the fusion protein is obtained by connecting the N end and/or the C end of the protein with the amino acid sequence shown as SEQ ID NO. 4 with a label;

i) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of an amino acid sequence shown as SEQ ID NO. 4;

g) protein with 75% or more than 75% homology with amino acid sequence shown as SEQ ID NO. 4 and the same function.

In a second aspect of the invention, there is provided the use of a nucleic acid molecule encoding a lipid transfer protein of rice as described above, OsLTP10 protein and OsLTP24 protein, for increasing the fatty acid content of rice or for reducing the chalkiness of rice.

In some embodiments of the present invention, the nucleic acid molecule encoding the OsLTP10 protein has the following nucleotide sequence:

(1) 1 or a degenerate sequence thereof;

(2) a nucleotide sequence which is derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined in the step (1), and codes a polypeptide with the same or similar function with the sequence of SEQ ID NO. 1. Namely, the unsaturated fatty acid content of the rice can be improved.

In some embodiments of the present invention, the above (2) is: the nucleotide sequence obtained by substituting, deleting or adding 1 or several (within 10) nucleotides in the nucleotide sequence defined in (1), and the nucleotide sequence codes a polypeptide with the same or similar functions with the sequence of SEQ ID NO. 1.

In some embodiments of the present invention, the nucleic acid molecule encoding the OsLTP24 protein has the following nucleotide sequence:

1) 3 or a degenerate sequence thereof;

2) a nucleotide sequence which is derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence limited by 1), and encodes a polypeptide with the same or similar function with the sequence of SEQ ID NO. 3.

In some embodiments of the present invention, the above 2) is: a nucleotide sequence which is derived by substituting, deleting or adding 1 or a plurality of (within 10) nucleotides in the nucleotide sequence limited by 1), and codes a polypeptide with the same or similar function with the sequence of SEQ ID NO. 3.

In a third aspect of the present invention, there is provided a biomaterial related to the above-mentioned nucleic acid molecule, which is any one of the following 1) to 7):

1) an expression cassette comprising the nucleic acid molecule;

2) a recombinant vector comprising the nucleic acid molecule;

3) a recombinant vector comprising 1) the expression cassette;

4) a recombinant microorganism containing the above-mentioned nucleic acid molecule;

5) a recombinant microorganism comprising 1) said expression cassette;

6) a recombinant microorganism containing 2) the recombinant vector;

7) a recombinant microorganism comprising 3) the recombinant vector.

In some embodiments of the invention, the nucleic acid molecule is a nucleic acid molecule encoding an OsLTP10 protein or a nucleic acid molecule encoding an OsLTP24 protein.

In a fourth aspect of the present invention, a primer set for amplifying a nucleic acid molecule encoding the above-mentioned OsLTP10 protein or a nucleic acid molecule encoding the above-mentioned OsLTP24 protein is provided.

In some embodiments of the invention, the primer set comprises a forward primer sequence as shown in SEQ ID NO. 5 and a reverse primer sequence as shown in SEQ ID NO. 6 or a forward primer sequence as shown in SEQ ID NO. 7 and a reverse primer sequence as shown in SEQ ID NO. 8.

In a fifth aspect of the present invention, a method of growing a high fatty acid content plant is presented, the method comprising the steps of: introducing the nucleic acid molecule encoding OsLTP10 protein, the nucleic acid molecule encoding OsLTP24 protein or the biological material into a receptor plant, and screening to obtain a transgenic plant.

In some embodiments of the invention, the method further comprises the step of transforming plant callus by agrobacterium-mediated method using an expression vector comprising a nucleic acid molecule encoding OsLTP10 protein and a nucleic acid molecule encoding OsLTP24 protein, and then growing the transformed plant into a transgenic plant.

In some embodiments of the invention, the plant is rice, arabidopsis, or canola.

In a sixth aspect of the present invention, a method of reducing the chalkiness rate of rice is proposed, said method comprising the steps of: and (3) introducing the nucleic acid molecule coding the OsLTP10 protein, the nucleic acid molecule coding the OsLTP24 protein or the biological material into receptor rice, and screening to obtain transgenic rice.

According to the embodiment of the invention, at least the following beneficial effects are achieved: the invention discloses application of rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness degree of rice, and rice mutants of OsLTP10 protein or OsLTP24 are knocked out to show that the fatty acid content is reduced, the chalkiness of grains is increased, and the rice quality is poor; and the chalkiness of over-expression strain seeds is obviously reduced, the content of fatty acid is increased, and the rice quality is improved, which shows that the OsLTP10 protein and the OsLTP24 protein have important functions for improving the rice quality. The invention provides a new thought for the research of molecular mechanism of rice chalkiness and fatty acid content and also provides a new gene resource for improving rice quality.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a map of pYLCRISPR vector in example 2 of the present invention;

FIG. 2 is a diagram of agarose gel electrophoresis detection results of PCR products of transgenic lines in example 2 of the present invention, wherein M is marker, and 1-6 are transgenic lines, respectively;

FIG. 3 is a diagram of agarose gel electrophoresis detection results of PCR products of transgenic lines in example 2 of the present invention, wherein M is marker, and 1-11 are transgenic lines, respectively;

FIG. 4 is a plasmid map of pCAMBIA1301 in example 3 of the present invention;

FIG. 5 is a diagram of agarose gel electrophoresis detection results of PCR products of transgenic lines in example 3 of the present invention, wherein M is marker, and 1-11 are transgenic lines, respectively;

FIG. 6 is a diagram showing the result of agarose gel electrophoresis detection of a PCR product of a transgenic line in example 3 of the present invention, wherein M is marker, and 1-9 are transgenic lines, respectively;

FIG. 7 is a graph showing peaks of fatty acid contents of rice in test examples according to the present invention;

fig. 8 is a phenotype diagram of mature seeds and their chalkiness of wild type zhenshan 97B rice, zhenshan 97B rice with the OsLTP10 gene knocked out, and zhenshan 97B rice with the OsLTP10 gene overexpressed in a test example of the present invention;

fig. 9 is a phenotype diagram of mature seeds and chalkiness of wild type zhenshan 97B rice, zhenshan 97B rice with the OsLTP24 gene knocked out, and zhenshan 97B rice with the OsLTP24 gene overexpressed in a test example of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1 Rice lipid transfer protein and Gene encoding the same

1. The OsLTP10 protein is shown as SEQ ID NO. 2, and the nucleotide sequence of the corresponding gene is shown as SEQ ID NO. 1.

The OsLTP10 gene corresponds to a sequence:

5’-ATGGCCCGTGCACAGTTGGTGTTGGTCGCCGTGGTGGCAGCTCTGCTCCTCGCCGCCCCGCACGCCGCCGTGGCCATCACCTGCGGCCAGGTGAACTCAGCCGTTGGCCCCTGCCTCACCTACGCCCGCGGCGGCGGCGCCGGCCCTTCCGCGGCCTGCTGCAACGGCGTGAGGAGCCTCAAGTCCGCTGCCAGAACCACCGCTGACCGGCGCACCGCCTGCAACTGCCTCAAGAACGCCGCCCGTGGCATCAAGGGGCTCAACGCCGGCAACGCCGCCAGCATCCCCTCCAAGTGCGGCGTCAGCGTCCCCTACACCATCAGCGCTTCCATCGACTGCTCTAGGGTGAGGTGA-3’(SEQ ID NO:1)。

OsLTP10 protein corresponding sequence:

MARAQLVLVAVVAALLLAAPHAAVAITCGQVNSAVGPCLTYARGGGAGPSAACCNGVRSLKSAARTTADRRTACNCLKNAARGIKGLNAGNAASIPSKCGVSVPYTISASIDCSRVR*(SEQ ID NO:2)。

2. the OsLTP24 protein is shown as SEQ ID NO. 4, and the nucleotide sequence of the corresponding gene is shown as SEQ ID NO. 3.

The OsLTP24 gene corresponds to the sequence:

5’-ATGGATGAGCACACAACACACACAAGAAAGAAGAAGAAGAAGAAGACGACGGCGAAGATGAAGGGAGCGGCTGCGGTGGCGATGGTGGTGGTGGCCGGCTGCTTGCTGGCGGCGGCGGCGGTGTCCGTCGTCGACGGCGCGGTGACGTGCGGCGACGTGGACGCGAGCCTGCTGCCGTGCGTGGCGTACCTGACCGGCAAGGCGGCGGCGCCCTCGGGGGATTGCTGCGCCGGCGTGAGGCACCTCCGGACGCTGCCCGTCGGCACCGCCGAGCGGCGGTTCGCCTGCGACTGCGTGAAGAAGGCGGCGGCGCGGTTCAAGGGGCTCAACGGCGACGCCATCCGCGACCTCCCGGCCAAGTGCGCCGCGCCGCTGCCGTTCCCGCTCAGCCTCGACTTCGACTGCAACACCATTCCATGA-3’(SEQ ID NO:3)。

the OsLTP24 protein corresponds to the sequence:

MDEHTTHTRKKKKKKTTAKMKGAAAVAMVVVAGCLLAAAAVSVVDGAVTCGDVDASLLPCVAYLTGKAAAPSGDCCAGVRHLRTLPVGTAERRFACDCVKKAAARFKGLNGDAIRDLPAKCAAPLPFPLSLDFDCNTIP*(SEQ ID NO:4)。

example 2 construction of knockout vector for Rice lipid transfer protein and transgene detection

Construction of knockout vector of A OsLTP10 gene and transgene detection

1. Gene editing site design

In this example, according to the existing CRISPR/Cas 9-related experimental method, 2 specific targets are designed according to the nucleotide sequence of OsLTP10, which are "GTAGGTGAGGCAGGGGCCAACGG" using CGG as a recognition site and "CGTTGCAGCAGGCCGCGGAAGGG" using GGG as a recognition site as a knockout target site, respectively, and it is expected that a loss-of-function transgenic plant is obtained to define the biological function of the OsLTP10 gene.

CRISPR/Cas9 vector construction

The CRISPR/Cas9 vector used in this example is pYLCRISPR (from the national emphasis laboratory for subtropical agriculture biological resource protection and utilization of agricultural resources at southern China university), the CRISPR/gRNA vectors map is shown in FIG. 1, and a target sequence is loaded by using a vector construction kit to form a recombinant vector containing an OsLTP10 gene target site. The specific operation method comprises the following steps:

(1) preparing a dimer: taking 1 mu L of primers before and after 10 mu M target site, adding 18 mu L of 0.5x TE, gently mixing, annealing in a PCR instrument at 99 ℃ for 3min, and naturally cooling to obtain a double-stranded sequence containing the knocked-out target site;

(2) ligation of dimer to pYLCRISPR: according to a reaction system of 1 mu L of dimer, 2 mu L of enzyme 1 mu L, CRISPR/Cas9 carrier and 2 mu L of ultrapure water, the components are mixed uniformly and reacted for 1h at 20 ℃ to complete sequence connection.

3. Transformation of E.coli

(1) And (3) transformation: taking out the competence of the escherichia coli cell T1 from-80 ℃, then unfreezing the escherichia coli cell T1 on ice, quickly adding the ligation product in the previous step after the cell is dissolved, gently mixing the cells by using a pipette gun, standing the mixture on ice for 30min, then thermally shocking the mixture at 42 ℃ for 30s, standing the mixture on ice for 2min again, then adding 10 times of LB (Luria Bertani) nonreactive culture solution into the converted product, and culturing the mixture at 37 ℃ and 200rpm for 50 min;

(2) coating a plate: taking out the activated bacterial liquid, centrifuging at 4000rpm, removing most of supernatant, coating the residual liquid (about 100 mu L) on a plate (LB + hygromycin resistance) after resuspension, and standing and culturing at 37 ℃ overnight;

(3) PCR detection of bacterial liquid: 3mL (LB + hygromycin resistance) of the monoclonal colony is picked for enlarged culture, a positive clone is selected, a plasmid is extracted, and the plasmid is sent to Shanghai biological engineering Limited company for sequencing confirmation.

4. Agrobacterium transformation

(1) And (3) transformation: taking out agrobacterium tumefaciens EHA105 cell competence (prepared by a chemical transformation method) from-80 ℃, then placing the cell competence on ice for thawing, adding 1 mu L of positive clone plasmid extracted in the step 3, placing the cell competence on ice for 30min after being gently mixed, then freezing the cell competence in liquid nitrogen for 2min, quickly taking out the cell competence, placing the cell competence on water bath at 37 ℃ for 2min, then adding 10 times of LB nonreactive culture solution into a transformation product, and culturing the cell competence at 28 ℃ and 250rpm for 2-3 h;

(2) coating a plate: taking out activated bacteria liquid, centrifuging at 5000rpm, removing most of supernatant, re-suspending the bacteria with residual liquid (about 100 μ L), plating (LB + rifampicin + hygromycin), and standing at 28 deg.C overnight for 36-48 h;

(3) detecting and shaking bacteria: picking the single colony, obtaining positive clone by sequencing detection, adding the positive colony with correct sequencing into a YEB liquid culture medium containing hygromycin, culturing overnight at 28 ℃, 200-250rpm until the OD600 value is about 0.8, centrifuging at 4000-5000rpm, then discarding the supernatant, and resuspending the precipitate by using an N6 liquid culture medium to obtain the recombinant agrobacterium tumefaciens.

5. Genetic transformation of rice

(1) Inducing callus: removing glumes of Zhenshan 97B rice, sterilizing with 3% sodium hypochlorite and 70% ethanol, and placing in a container containing 2.5mg.L ^-1 2,4-D on N6 solid medium, inducing for 10-14 days to obtain embryogenic callus;

(2) infection of agrobacterium: peeling the induced embryogenic callus from the explant by using a scalpel, infiltrating the selected callus in the environment of the resuspended recombinant agrobacterium tumefaciens prepared in the step 3 for about 30min, transferring the callus to sterile filter paper, sucking water, and transferring the callus to an N6 culture medium containing 100TM acetosyringone for culture for 3 days;

(3) screening: cleaning callus with sterile water, transferring into a container containing 25-50mg.L ^-1 Hygromycin and 200-400mg ^-1 Screening on N6 solid culture medium containing sodium carboxyvariegated penicillin for 1-3 weeks, transferring the resistant callus into new culture medium, screening for 2-3 weeks, transferring the resistant callus into MS culture medium, differentiating, and culturing to obtain transformed plant (T) ₁ Generation transformed plant) and then transplanting in a pot to carry out conventional technology for rice cultivation management.

(4) T cultivated from pots ₁ Removing rice leaves of a generation transformation plant according to a single plant, extracting a DNA sample by adopting a CTAB method (a hexadecyl trimethyl ammonium bromide method), and carrying out PCR detection analysis by using primers 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' (SEQ ID NO:9) and 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' (SEQ ID NO:10) corresponding to a hygromycin resistance gene sequence, wherein the reaction system is as follows: 10uM of each of the upstream and downstream primers, 1. mu.L of DNA template, 11. mu.L of 2 XPCR premix, and 7. mu.L of double distilled water, wherein the total amount is 20. mu.L; the PCR reaction program is: 95 ℃ 20s, 58 ℃ 20s, 72 ℃ 30s, 35 cycles. The result of electrophoresis gel running of the PCR product is shown in FIG. 2, and the one capable of detecting a positive band is T ₁ And (4) generating transgenic lines, and collecting seeds according to single plants for subsequent tests.

Construction of knockout vector of B OsLTP24 gene and transgene detection

1. Gene editing site design

In this example, according to the existing CRISPR/Cas 9-related experimental method, 2 specific targets are designed according to the nucleotide sequence of OsLTP24, which are "gacggcgaagatgaagggagcg" using CGG as a recognition site and "ccgtgcgtggcgtacctgaccgg" using GGG as a recognition site as a knockout target site, respectively, and it is expected that a loss-of-function transgenic plant is obtained to define the biological function of the OsLTP24 gene.

CRISPR/Cas9 vector construction

The CRISPR/Cas9 vector is used as pYLCRISPR, and a vector construction kit is used for loading a target sequence to form a recombinant vector containing the OsLTP24 gene target site. The specific operation method comprises the following steps:

(1) preparation of dimer: taking 1 mu L of primers before and after 10 mu M target site, adding 18 mu L of Buffer Aneal, gently mixing, annealing in a PCR instrument at 99 ℃ for 3min, and naturally cooling to obtain a double-stranded sequence containing the knocked-out target site;

3. Transformation of E.coli

(3) PCR detection of bacterial liquid: selecting a monoclonal colony for expanding culture for 3mL (LB + hygromycin resistance), selecting a positive clone, extracting a plasmid, and sending the plasmid to Shanghai biological engineering Limited company for sequencing confirmation.

4. Agrobacterium transformation

(3) detecting and shaking bacteria: selecting a monoclonal colony, obtaining a positive clone by sequencing detection, adding the positive colony with correct sequencing into a YEB liquid culture medium containing hygromycin, carrying out overnight culture at 28 ℃, 200-5000 rpm until the OD600 value is about 0.8, centrifuging at 4000-5000rpm, then removing a supernatant, and carrying out heavy suspension precipitation by using an N6 liquid culture medium to obtain the recombinant agrobacterium tumefaciens.

5. Genetic transformation of rice

(4) T cultivated from pots ₁ Removing rice leaves of a generation transformation plant according to a single plant, extracting a DNA sample by adopting a CTAB method (a hexadecyl trimethyl ammonium bromide method), and carrying out PCR detection analysis by using primers 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' (SEQ ID NO:9) and 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' (SEQ ID NO:10) corresponding to a hygromycin resistance gene sequence, wherein the reaction system is as follows: 10uM of each upstream and downstream primer 1 uL, 1 uL of DNA template, 11 uL of 2 XPCR premix and 7 uL of double distilled water, and the total amount is 20 uL; the PCR reaction program is: 95 ℃ 20s, 58 ℃ 20s, 72 ℃ 30s, 35 cycles. The result of PCR product after running gel is shown in FIG. 3, and the positive band detected is T ₁ And (4) generating transgenic lines, and collecting seeds according to single plants for subsequent tests.

Example 3 construction of overexpression vector and transgene detection of Rice lipid transfer protein in Rice

Overexpression vector construction and transgene detection of OsLTP10 gene in A rice

1. Overexpression vector construction

Extracting pCAMBIA1301 plasmid In DH5 alpha by using a plasmid extraction kit, wherein the map of the pCAMBIA1301 plasmid is shown In figure 4, carrying out BamHI and Kpnl double enzyme digestion on the plasmid respectively, wherein a double enzyme digestion system is shown In table 2, transforming a nucleotide sequence shown In SEQ ID No.1 into the pCAMBIA1301 plasmid after double enzyme digestion, connecting the pCAMBIA1301 plasmid with an In-Fusion kit of TaKaRa company, connecting an OsLTP10 gene fragment with the pCAMBIA1301 plasmid subjected to double enzyme digestion, and carrying out reaction assembly at 50 ℃ for 15 min. Spread on LB agar medium containing 30. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain pCAMBIA1301-OsLTP 10. PCR identification was performed using primers OsLTP10-F and OsLTP10-R, the nucleotide sequences of which are shown in Table 1, and BamHI-digested positive plasmids were sent to Shanghai Bioengineering Co., Ltd for sequencing verification.

TABLE 1

TABLE 2 double restriction system for plasmids

2. Transformation of E.coli

(1) And (3) transformation: taking out escherichia coli cell DH5 alpha competence (purchased from Tiangen biochemistry Co., Ltd.) from-80 ℃, then placing on ice for thawing, quickly adding the ligation product in the last step after the cell is dissolved, gently mixing the cells by using a pipette, then standing the mixture on ice for 30min, then carrying out heat shock at 42 ℃ for 30s, standing the mixture on ice for 2min again, then adding 10 times of LB (Lubtree) culture solution into the transformation product, and culturing the transformation product at 37 ℃ and 200rpm for 50 min;

(2) coating a plate: taking out the activated bacterial liquid, centrifuging at 4000rpm, removing most of supernatant, re-suspending the residual liquid (about 100 mu L), coating a plate (LB + kanamycin resistance), and standing and culturing at 37 ℃ overnight;

(3) PCR detection of bacterial liquid: the next day, 3mL (LB + kanamycin resistance) of the single colony is picked for enlarged culture, a positive clone is selected and a plasmid is extracted, and the plasmid is sent to Shanghai biological engineering company Limited for sequencing confirmation.

3. Agrobacterium transformation

(1) And (3) transformation: taking out agrobacterium tumefaciens EHA105 cell competence (prepared by a chemical transformation method) from-80 ℃, then placing the cell competence on ice for thawing, adding 1 mu L of prepared positive clone plasmid, placing the cell competence on ice for 30min after being gently mixed, then freezing the cell competence in liquid nitrogen for 2min, quickly taking out the cell competence, placing the cell lyse in 37 ℃ water bath for 2min, then adding 10 times of LB nonreactive culture solution into the transformation product, and culturing the cell for 2-3h at 28 ℃ and 250 rpm;

(2) coating a plate: taking out activated bacteria liquid, centrifuging at 5000rpm, removing most of supernatant, re-suspending the bacteria with residual liquid (about 100 μ L), plating (LB + rifampicin + kanamycin), and standing at 28 deg.C overnight for 36-48 h;

(3) detecting and shaking bacteria: selecting a monoclonal colony, screening positive clones by using a culture medium containing kanamycin and rifampicin, adding the positive colony into a YEB liquid culture medium containing kanamycin, culturing at 28 ℃, 200-5000 rpm overnight until the OD600 value is about 0.8, centrifuging at 4000-5000rpm, then discarding the supernatant, and carrying out heavy suspension precipitation by using an N6 liquid culture medium to obtain the recombinant agrobacterium.

4. Genetic transformation of rice

(2) and (3) agrobacterium infection: peeling the induced embryogenic callus from the explant by using a scalpel, infiltrating the selected callus for about 30min in the environment of the resuspended recombinant agrobacterium tumefaciens prepared in the step 3, transferring the callus to sterile filter paper, sucking water, and transferring the callus to an N6 culture medium containing 100TM acetosyringone for culture for 3 days;

(3) screening: cleaning callus with sterile water, transferring into a container containing 25-50mg.L ^-1 Hygromycin and 200-400mg ^-1 Screening on N6 solid culture medium of carboxyvaricin sodium for 1-3 weeks, transferring the resistant callus into new culture medium, further screening for 2-3 weeks, transferring the resistant callus into MS culture medium, differentiating, and rooting culturing to obtain transformed plant (T) ₁ Generation transformed plant) and then transplanting in a pot to carry out conventional technology for rice cultivation management.

(4) T cultivated from pots ₁ Removing rice leaves of a generation transformation plant according to a single plant, extracting a DNA sample by adopting a CTAB method (a hexadecyl trimethyl ammonium bromide method), and carrying out PCR detection analysis by using primers 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' (SEQ ID NO:9) and 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' (SEQ ID NO:10) corresponding to hygromycin resistance gene sequences, wherein the reaction system is as follows: 10uM of each of the upstream and downstream primers, 1. mu.L of DNA template, 101. mu.L of 2 XPCR premix, and 7. mu.L of double distilled water, wherein the total amount is 20. mu.L; the PCR reaction program is: 95 ℃ 20s, 58 ℃ 20s, 72 ℃ 30s, 35 cycles. The result graph after electrophoresis gel-running of the PCR product is shown in FIG. 5, and the one capable of detecting a positive band is T ₁ Generation of transgenic lines, transformation of T ₁ The generation transgenic line collects seeds according to single plants for subsequent tests.

B overexpression vector construction and transgene detection of OsLTP24 gene in rice

1. Overexpression vector construction

Extracting pCAMBIA1301 plasmid In DH5 alpha by using a plasmid extraction kit, carrying out double enzyme digestion on the plasmid respectively with BamHI and Kpnl, wherein the double enzyme digestion system is shown In Table 2, transforming the nucleotide sequence shown In SEQ ID No.3 into the pCAMBIA1301 plasmid after double enzyme digestion, connecting the pCAMBIA1301 plasmid by using an In-Fusion kit of TaKaRa company, connecting an OsLTP24 gene fragment with the pCAMBIA1301 plasmid subjected to double enzyme digestion, and carrying out reaction assembly at 50 ℃ for 15 min. Spread on LB agar medium containing 30. mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain pCAMBIA1301-OsLTP 24. PCR was performed using primers OsLTP24-F and OsLTP24-R, the nucleotide sequences of which are shown in Table 3, and BamHI-digested positive plasmids were subjected to sequencing verification by Shanghai Bioengineering Co., Ltd.

TABLE 3

2. Transformation of E.coli

3. Agrobacterium transformation

(3) detecting and shaking bacteria: selecting a monoclonal colony, screening positive clones by using a culture medium containing kanamycin and rifampicin, adding the positive colony into a YEB liquid culture medium containing kanamycin, culturing overnight at 28 ℃, 200-5000 rpm until the OD600 value is about 0.8, centrifuging at 4000-5000rpm, discarding a supernatant, and re-suspending and precipitating by using an N6 liquid culture medium to obtain the recombinant agrobacterium.

4. Genetic transformation of rice

(3) screening: cleaning callus with sterile water, transferring into a container containing 25-50mg.L ^-1 Hygromycin and 200-400mg ^-1 Screening on N6 solid culture medium containing sodium carboxyvariegated penicillin for 1-3 weeks, transferring the resistant callus into new culture medium, screening for 2-3 weeks, transferring the resistant callus into MS culture medium, differentiating, and culturing to obtain transformed plant (T) ₁ Generation transformed plants) and then transplanting in pots for conventional technology to carry out rice cultivation management.

(4) T cultivated from pots ₁ Removing rice leaf from the transformed plant according to a single plant, extracting DNA sample by CTAB method (cetyl trimethyl ammonium bromide method), and using the carried hygromycin resistance gene sequencePCR assay was performed with primers 5'-ACGGTGTCGTCCATCACAGTTTGCC-3' (SEQ ID NO:9) and 5'-TTCCGGAAGTGCTTGACATTGGGGA-3' (SEQ ID NO:10) corresponding to the columns in the reaction system: 10uM of each upstream and downstream primer 1 uL, 1 uL of DNA template, 11 uL of 2 XPCR premix and 7 uL of double distilled water, and the total amount is 20 uL; the PCR reaction program is: 95 ℃ 20s, 58 ℃ 20s, 72 ℃ 30s, 35 cycles. FIG. 6 shows the result of electrophoresis of PCR products, where T is the positive band detected ₁ And (4) generating transgenic strains, and collecting seeds according to single plants for subsequent tests.

Test examples

The experimental method comprises the following steps: adopting field test, dividing wild Zhenshan 97B rice after removing OsLTP10 gene, Zhenshan 97B rice after removing OsLTP24 gene, Zhenshan 97B rice after over-expressing OsLTP10 gene and Zhenshan 97B rice after over-expressing OsLTP24 gene into 20 test fields respectively, each group is 4, planting alone, managing in normal field, placing mature and harvested rice seeds in a 37 ℃ oven for 5D after harvesting rice, drying, mechanically removing husk, beating into polished rice by a polished rice machine, analyzing fatty acid content and component difference in rice by gas chromatography-mass spectrometry (GC-MS), and determining rice chalkiness degree by micro-CT and 3D reconstruction technologies.

1. Fatty acid content detection

And respectively analyzing the content and component difference of fatty acids in the harvested wild Zhenshan 97B rice, the Zhenshan 97B rice with the OsLTP10 gene knocked out, the Zhenshan 97B rice with the OsLTP24 gene knocked out, the Zhenshan 97B rice seed with the OsLTP10 gene overexpressed and the Zhenshan 97B rice seed with the OsLTP24 gene overexpressed by a gas chromatography-mass spectrometry (GC-MS) method. Each group of material was randomly ground to powder at 30 grains of rice.

After derivatization treatment, the sample is used for determining the content of fatty acid and the ratio of each fatty acid component by a GC-MS method, and the method comprises the following steps: weighing 0.2g of powder into a10 mL test tube for derivatization treatment of one-step acid catalytic methylation, adding 5mL of methyl sulfate, and placing at 60 ℃ for acid ester derivatization treatment for 8 hours; and after the reaction is finished, adding 2mL of n-hexane, uniformly mixing and extracting, centrifuging at a high speed for 1 minute, taking the supernatant, filtering, injecting the supernatant into a gas chromatograph-mass spectrometer for analysis, checking each peak with a standard spectrum library through mass spectrometry and a known compound control method, confirming 12 fatty acid components, and calculating the content of the fatty acid.

GC-MS measurement conditions:

chromatographic conditions carrier gas: SH-Rt-2560 column of 100m × 0.25mm × 0.25 μm, sample introduction temperature of 240 deg.C, and sample loading of 1 microliter. The gasification temperature is 260 ℃, the split ratio is 10: 1, helium (99.999%) is taken as carrier gas, the flow rate is 1mL/min, the initial column temperature is 150, the temperature is increased to 180 ℃ at 10 ℃/min, the temperature is maintained for 2min, then the temperature is increased to 200 ℃ at 3 ℃/min, the temperature is maintained for 20min, and finally the temperature is increased to 230 ℃ at 0.5 ℃/min.

Mass spectrometry conditions EI ion sources; ionization energy 70 eV; ion source temperature: 280 ℃; temperature of the quadrupole rods: 150 ℃; transmission line temperature: 290 ℃; multiplier voltage: 2000V; scanning mass range: 30-450 amu; GC-MS interface temperature: 230 ℃ to 230 ℃.

TABLE 4

Line of plants	Fatty acid content (mg/g)	Content variation ratio (average value)
			Wild type (ZS97B)	32.65	-
OsLTP10 overexpression strain	34.52	5.72％
			OsLTP10 knockout strain	29.63	-9.23％
OsLTP24 overexpression strain	34.88	6.81％
			OsLTP24 knockout strain	30.09	-7.82％

TABLE 5

Note: ZS97B is Zhenshan 97B; OE-LTP10 and OE-LTP24 are OsLTP10 and OsLTP24 overexpression strains respectively; CR-LTP10 and CR-LTP24 are OsLTP10 and OsLTP24 gene knockout strains respectively.

As shown in Table 4, Table 5 and FIG. 7, it can be seen from Table 4 that the fatty acid content of the overexpression strain of OsLTP10 gene was increased by 5.72% compared with the wild type in the overexpression strain of OsLTP10 gene; the number of the lines with the OsLTP10 gene knocked out is reduced by 9.23%; compared with the wild type, the relative content of fatty acid in the overexpression strain of the OsLTP24 gene is increased by 6.81 percent; the number of lines of the OsLTP10 gene knockout strain is reduced by 7.82 percent; the results of comparing the fatty acid component contents of different transgenic lines of OsLTP10, different lines of which OsLTP10 gene is knocked out and wild type Zhenshan 97B are shown in Table 5, and it can be seen from Table 5 that the over-expression of OsLTP10 gene can reduce the linoleic acid (C18:2) ratio by about 3.7% and increase the oleic acid (C18:1) ratio by about 8.2%, while the lines of which OsLTP10 gene is knocked out show that the linoleic acid change is not obvious, but the oleic acid (C18:1) ratio is reduced by about 4.9%, and the change of the expression of OsLTP24 gene has the same effect on the fatty acid component. Fig. 7 is a peak diagram of the content of each component of fatty acid in rice, and as can be seen from fig. 7, table 4 and table 5, rice varieties with high oleic acid and low linoleic acid can be obtained by regulating and controlling OsLTP10 and OsLTP24, thereby improving the quality of rice.

2. Detection of chalkiness degree of rice

The results of rice whiteness comparison experiments of wild type Zhenshan 97B rice, Zhenshan 97B rice after removing OsLTP10 gene, Zhenshan 97B rice after removing OsLTP24 gene, Zhenshan 97B rice after over-expressing OsLTP10 gene and Zhenshan 97B rice after over-expressing OsLTP24 gene are shown in FIGS. 8-9, and it can be seen from FIG. 8 that the whiteness of rice in OsLTP10 over-expressed strain is significantly reduced, the whiteness of rice in gene-knocked out OsLTP10 strain (OsLTP10 gene edited strain) is significantly increased, and from FIG. 9, compared with wild type, the whiteness of OsLTP24 over-expressed strain is significantly reduced, while the whiteness of rice in gene-knocked out OsLTP24 strain (OsLTP24 gene edited strain) is significantly increased, and the effect of OsLTP24 gene expression on OsLTP10 is consistent with that of OsLTP.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

Sequence listing

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Claims

1. The application of the rice lipid transfer protein in improving the fatty acid content of rice and reducing the chalkiness degree of the rice is characterized in that the rice lipid transfer protein is OsLTP10 protein and OsLTP24 protein.

2. The use according to claim 1, wherein the OsLTP10 protein is a protein of a) or b) or c) or d) as follows:

a) protein with amino acid sequence shown as SEQ ID NO. 2;

b) the fusion protein is obtained by connecting a label to the N end and/or the C end of the protein with the amino acid sequence shown as SEQ ID NO. 2;

3. The use according to claim 1, wherein the OsLTP24 protein is a protein of e) or f) or h) or i) as follows:

e) protein with amino acid sequence shown as SEQ ID NO. 4;

4. Use of a nucleic acid molecule encoding a rice lipid transfer protein as claimed in claim 1 for increasing the fatty acid content of plant seeds and for reducing the chalkiness of rice, wherein said rice lipid transfer protein is OsLTP10 protein and OsLTP24 protein.

5. The use as claimed in claim 4, wherein the nucleic acid molecule encoding OsLTP10 protein has the following nucleotide sequence:

(1) 1 or a degenerate sequence thereof;

(2) a nucleotide sequence which is derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined in the step (1), and codes a polypeptide with the same or similar function with the sequence of SEQ ID NO. 1.

6. The use as claimed in claim 4, wherein the nucleic acid molecule encoding OsLTP24 protein has the following nucleotide sequence:

1) 3 or a degenerate sequence thereof;

7. Primer set for amplifying a nucleic acid molecule encoding OsLTP10 protein as claimed in claim 4 or 5, or a nucleic acid molecule encoding OsLTP24 protein as claimed in claim 4 or 6.

8. The primer set of claim 7, wherein the primer set comprises a forward primer sequence shown as SEQ ID NO. 5 and a reverse primer sequence shown as SEQ ID NO. 6 or a forward primer sequence shown as SEQ ID NO. 7 and a reverse primer sequence shown as SEQ ID NO. 8.

9. A method of growing plants with high fatty acid content, comprising the steps of: introducing a nucleic acid molecule coding OsLTP10 protein or a nucleic acid molecule coding OsLTP24 protein into a receptor plant, and screening to obtain a transgenic plant; preferably, the plant is rice, arabidopsis thaliana or oilseed rape.

10. A method of reducing the chalkiness rate of rice, said method comprising the steps of: and (3) introducing the nucleic acid molecule coding the OsLTP10 protein or the nucleic acid molecule coding the OsLTP24 protein into receptor rice, and screening to obtain the transgenic rice.