CN117866983B - Application of OsbZIP10 gene in regulation of amylose content of rice grains - Google Patents
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Abstract
The invention discloses an application of an OsbZIP10 gene in regulating and controlling the amylose content of rice grains. The nucleotide sequence of the protein coding region of the OsbZIP10 gene is shown as SEQ ID NO. 1. The novel application of the OsbZIP10 gene in reducing the amylose of rice grains is discovered by knocking out the OsbZIP10 gene in common rice, and important gene resources are provided for improving the rice quality.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsbZIP10 gene in regulation and control of rice grain amylose content.
Background
With the improvement of the living standard of people, the improvement of rice quality has become a new pursuit of the common people, and is helpful for attracting consumers and promoting consumption (Tian et al, 2009). For example, rice germplasm with a low Amylose Content (AC) has the amylose content of the endosperm of a larger consumer audience (Huang et al 2020) as a major factor affecting rice quality. The low-amylose rice with the amylose content of 5-15% has the characteristics of soft rice, oily and glossy appearance, no retrogradation after cooling, good puffing property and the like, and not only becomes the excellent rice directly cooked by people, but also is a superior raw material for processing instant cooked rice, puffed food, rice desserts and the like. Therefore, the search for high-yield and high-quality rice varieties is important for rice breeding.
Starch biosynthesis is a complex biological process, determined by the synergistic effects of various factors, such as carbon partitioning, phytohormones, transcription Factors (TF), redox homeostasis, etc. (Ma et al, 2023). Changes in metabolic flux are responsible for starch granule formation, such as carbon metabolism (Baysal et al, 2020) and amino acid metabolism (Yan et al, 2022). Phytohormones are another important factor in regulating seed starch development, such as auxins (Paul et al, 2020), cytokinins (Hu et al, 2023), jasmonic acid (JA; hu et al, 2023) and abscisic acid (ABA; yang et al, 2020; 2022). In addition, reactive Oxygen Species (ROS) are now becoming an important focus of influencing grain yield and quality (Wu et al, 2022). It is also notable that regulation of seed formation is largely dependent on interactions between different factors. For example, rice OsGSA1 coordinates sugar metabolism and abiotic stress to control seed size (Dong et al 2020), and a synergistic effect between auxin homeostasis and carbon metabolism is responsible for MADS-mediated early seed development in rice (Paul et al 2020). Furthermore, programmed Cell Death (PCD) plays a vital role in starch formation and seed maturation, which may also be induced by ROS (Wu et al, 2022).
In addition, many transcription factors have been reported to affect grain quality and yield mainly by directly regulating genes involved in starch biosynthesis. OsbZIP58 (basic leucine zipper 58) controls the appearance of rice by directly modulating starch synthesis genes, GBSSI (particle-bound starch synthase i, also known as wall, wx), ssia (starch synthase IIa) and SBEI (starch branching enzyme i) (Wang et al, 2013), whereas two ethylene-related transcription factors, RSR1 (rice starch regulator 1) and SERF1 (salt-reactive ethylene response factor 1), appear to be inversely related to starch synthesis by inhibiting the expression of starch synthesis genes (such as Wx) (SCHMIDT ET al, 2014). Two MADS-box transcription factors play an important role in early seed development by regulating genes involved in starch synthesis and auxin transport (Paul et al 2020). More importantly, the synchronization or synergy between different transcription factors also plays a critical role in controlling grain filling. For example, zmABI, in cooperation with ZmABI29, directly regulates Opaque2 (Yang et al, 2022) in corn endosperm grout; the interaction between OsEBP and OsBP determines the expression of Wx in rice and further controls AC (Hu et al 2023). In addition, transcription factor mediated cascade signaling is also responsible for grain filling. Rice EIL1-ERF115 and GW2-WG1-OsbZIP47 regulation modules are disclosed to control rice grain size and weight (Hao et al, 2021; liu et al, 2022).
The bZIP transcription factor families all have a highly conserved bZIP domain of 60-80 amino acids. It is composed of two parts: highly conserved basic regions of binding DNA and variable leucine zipper regions (Wang et al, 2008). bZIP proteins are an important class of transcription factors that recognize cis-acting elements such as the A, C and G cassettes containing the ACGT motif, and bZIP-type transcription factors are usually post-translationally modified or dimerized to function (Schu tze et al., 2008). Many starch synthesis-related genes contain ABA responsive elements (ABREs) in their promoter sequences to which activated bZIP-like transcription factors can bind, thereby regulating expression of downstream genes. Rice (Oryza satival.) is an important food crop, osbZIP10 is a transcription factor of the rice bZIP family, and has been reported to be involved in abiotic stress response signaling of low temperature, salt and the like of rice, but has not been reported in the aspect of rice quality improvement.
Disclosure of Invention
The invention provides an application of an OsbZIP10 gene in regulating and controlling the amylose content of rice grains, and the OsbZIP10 gene provides a basis for improving the rice quality, and the specific technical scheme is as follows:
In a first aspect, the invention provides an application of an OsbZIP10 gene in regulating and controlling amylose content of rice grains, wherein the nucleotide sequence of a protein coding region of the OsbZIP10 gene is shown as SEQ ID NO.1, the length of the protein coding region is 1167 bp, and the whole gene sequence is shown as SEQ ID NO. 2.
Further, the regulation is to reduce the amylose content of rice grains by silencing the OsbZIP10 gene or to increase the amylose content of rice grains by overexpressing the OsbZIP10 gene.
In a second aspect, the invention provides application of a bZIP family transcription factor coded by a rice OsbZIP10 gene in regulation and control of rice grain amylose, the bZIP family transcription factor consists of 388 amino acids, and the amino acid sequence of the bZIP family transcription factor is shown as SEQ ID NO. 3.
Further, the bZIP family transcription factor increases the amylose content of rice by enhancing sucrose metabolism, lipid metabolism and active oxygen scavenging ability.
The invention uses CRISPR/Cas9 gene editing technology to knock out OsbZIP10 genes in japonica rice varieties Japanese (Nip, oryza. Sativa L. Spp. Japonica, var. Nipponbare), shanghai rice 68 (HD 68, oryza. Sativa L. Spp. Japonica, var. Hudao 68) and Zhonghua11 (ZH 11, oryza. Sativa L. Spp. Japonica, var. Zhonghua 11) to prepare OsbZIP10 gene knocked-out plants OsbZIP-1 and OsbZIP-2 (Nip background), osbZIP-1 HD68 and OsbZIP-2 HD68 (HD 68 background), osbZIP-1 ZH11 and OsbZIP-2 ZH11 (ZH 11 background).
According to the invention, the OsbZIP10 gene is used as a target gene to be introduced into japonica rice variety Nipponica (Nip, oryza. Sativa. Spp. Japonica) to obtain a T 0 generation over-expression plant of the OsbZIP10 gene, and continuous selfing is carried out to obtain a homozygous high-expression T 2 generation plant line which is named as OsbZIP10-ox1 and OsbZIP10-ox2.
By performing direct starch content measurement on the OsbZIP10 knock-out rice OsbZIP-1 and OsbZIP-2, the osbZIP10-1 HD68 and OsbZIP10-2 HD68,osbzip10-1ZH11 and OsbZIP10-2 ZH11, T2 generation over-expression homozygous lines of OsbZIP10-ox1 and OsbZIP10-ox2 and seeds of the wild type control materials thereof, the direct starch content of the OsbZIP10 knock-out rice seeds is found to be obviously lower than that of the control, and the direct starch content of the OsbZIP10 over-expression rice seeds is obviously higher than that of the control.
The expression levels of sucrose metabolic gene (OsBGLU) and lipid metabolic genes (OsLTP and OsOLE 18) and active oxygen scavenging gene (OsHSFC B) were detected by taking the snapping samples of rice of different materials 7 days after flowering. The result shows that compared with the sucrose metabolism (OsBGLU gene expression), lipid metabolism (OsLTP and OsOLE18 gene expression) and active oxygen removal capacity (OsHSFC B gene expression) of the control material, the OsbZIP10 gene knockout rice grain is obviously reduced.
The experiments above are summarized to prove that: the knockout of the OsbZIP10 gene can reduce the direct-connection starch content of rice grains. The bZIP transcription factor coded by the OsbZIP10 gene can enhance sucrose metabolism (OsBGLU gene expression), lipid metabolism (OsLTP and OsOLE18 gene expression) and active oxygen removal capacity (OsHSFC B gene expression), and improve the amylose content of rice.
In a third aspect, the invention provides an application of a related biological material of a rice gene OsbZIP10 in regulation of amylose content of rice seeds, wherein the related biological material comprises the following components: an over-expression vector or a genetically engineered bacterium, and a CRISPR/Cas9 vector or a genetically engineered bacterium for knocking out the OsbZIP10 gene.
In a fourth aspect, the present invention provides a method for regulating amylose content in rice kernels, the method comprising:
(1) The OsbZIP10 gene is knocked out, so that the amylose content in rice grains is reduced;
(2) Through over-expression of the OsbZIP10 gene, the amylose content in rice grains is improved.
Further, the method for knocking out the OsbZIP10 gene comprises the following steps:
(1) According to the OsbZIP10 genome sequence, designing target sequence sgRNA, and constructing a CRISPR/Cas9 vector edited by the OsbZIP10 gene;
(2) Transferring the vector into an agrobacterium competent cell, and constructing a genetic engineering bacterium containing a knock-out OsbZIP10 gene CRISPR/Cas9 vector;
(3) Culturing the agrobacterium engineering bacteria to mediate transformation of rice callus to obtain a homozygous strain knocking out the OsbZIP10 gene.
Further, the nucleotide sequence of the target sequence sgRNA is shown as SEQ ID NO. 4.
Further, in the preparation process of the CRISPR/Cas9 vector in the step (1), an upstream primer is shown as SEQ ID NO.5, and a downstream primer is shown as SEQ ID NO. 6.
Further, the method for over-expressing the OsbZIP10 gene comprises the following steps:
(1) Designing a primer sequence by taking wild rice cDNA as a template, and constructing an overexpression vector of the OsbZIP10 gene;
(2) Transferring the vector into an agrobacterium competent cell to obtain agrobacterium for over-expressing the OsbZIP10 gene;
(3) Culturing the agrobacterium engineering bacteria to mediate transformation of rice callus to obtain a homozygous strain of the overexpression OsbZIP10 gene.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention discovers the new application of reducing the amylose of rice seeds by over-expressing and knocking out the OsbZIP10 gene in the plants of a plurality of rice varieties (Japanese sunny, shanghai rice 68 and Chinese flowering 11), and provides important gene resources for cultivating rice varieties with low amylose;
(2) The invention provides a breeding method of rice germplasm with low amylose by using CRISPR technology, and obtains OsbZIP10 gene knockout plants with low amylose.
Drawings
FIG. 1 shows amylose content of Oryza sativa and Oryza sativa kernels having undergone gene knockout and overexpression of OsbZIP10 in Japanese sunny province in example 3. a is the phenotype of mature grains in wild type, osbZIP10-1, osbZIP10-2, osbZIP10-ox1 and OsbZIP10-ox2, bar=8 mm. b is the phenotype of mature grains of different materials, bar=800 μm. c is PAS staining of mature grain paraffin sections, bar=800 μm. d is a Scanning Electron Microscope (SEM) image of mature kernels, bar=800 μm; red and blue squares represent the inner and outer regions, respectively. e is the central area of the grain in the d graph, bar=20μm. f is the peripheral area of the grain in the d graph, bar=20μm. g is the amylose content of the mature grain. h is the gum consistency in the mature grain. i is the gelatinization temperature in the mature grain. j is 1000 grain weight of mature grain.
FIG. 2 shows the amylose content of OsbZIP10 gene knockout and over-expression rice and ordinary rice grains of Shanghai rice 68 and Zhonghua 11 rice materials in example 3. a and b are phenotypes of mature grains in wild type, osbzip10-1 and osbzip10-2, bar=8 mm. c and d are the central and peripheral areas of the kernels in figures a and b, bar=20μm. e and h are the amylose content of the mature grain. f and i are the gum consistencies in the mature grain. g and j are gelatinization temperatures in the mature grain.
FIG. 3 shows the gene expression levels of the Nipponbzip 10 gene knockout and over-expression rice and ordinary rice grains in example 3. Transcriptional expression levels of sucrose metabolizing gene (OsBGLU), lipid metabolizing gene (OsLTP 2 and OsOLE) and active oxygen scavenging gene (OsHSFC 1B) at 7 days post flowering in different rice materials.
Detailed Description
In order to make the present invention more well understood by those skilled in the art, the following description of the present invention will be made with reference to specific embodiments. It should be noted that the following detailed description is exemplary and is merely an example of a portion, but not all, of the present invention.
All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The experimental materials used in the embodiment of the application are all conventional in the field and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the instructions recommended by the suppliers.
In the invention, the nucleotide sequence of a protein coding region of the OsbZIP10 gene is shown as SEQ ID NO.1, the full-length sequence of the OsbZIP10 gene is shown as SEQ ID NO.2, the protein coded by the OsbZIP10 gene is a bZIP family transcription factor and consists of 388 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO.3, and specifically comprises the following steps:
SEQ ID NO.1：
ATGGCATCGGAGATGAGCAAGAACGTGAAGGTCACCGATGATCAAGAGGTTACATCACAGGAGCGTGACCAAAGTGGTGGTACAAAAGTAGGTGGGGAGGAGGAAATTGCTCCACTGGCGCGGCAGTCGTCAATCCTCTCCCTCACCTTGGAAGAGCTACAAAACTCCTTGTGTGAGCCAGGACGCAACTTTGGTTCCATGAACATGGACGAGTTTGTGGCTAACATATGGAATGCTGAAGAATTCCAGGCTACCACCGGAGGTTGCAAGGGTGCCATGGAGGAAGCCAAGGTGGTAGACAGTGGAAGCGGAAGCGGTGATGCAGGAGGAAGCGGTTTATGTCGGCAGGGATCATTTTCCTTGCCGCTACCGCTGTGCCAGAAGACGGTGGAGGAGGTGTGGACTGAGATCAACCAAGCCCCTGCACACACCTCCGCTCCGGCCTCCGCGCTCCAGCCACATGCCGGGAGCGGTGGTGTTGCAGCTAACGACCGACAGGTAACACTAGGTGAGATGACACTTGAGGATTTCTTGGTAAAGGCCGGGGTGGTCCGAGGGTCCTTTACCGGGCAAGCGGCCATGGGATCTGGCATGGTCAACGGGCCGGTGAACCCCATGCAGCAGGGCCAAGGCGGTCCTATGATGTTCCCAGTAGGACCGGTAAACGCCATGTATCCGGTGATGGGTGATGGCATGGGGTACCCCGGTGGGTACAACGGGATGGCGATTGTGCCACCGCCACCTCCCGCCCAAGGTGCCATGGTTGTCGTGAGTCCTGGATCATCAGATGGGATGAGTGCCATGACACATGCTGATATGATGAATTGTATTGGGAATGGGATGATGATTGAGAATGGAACAAGAAAGCGTCCCCACAGAGAGGATGGCTGCGCCGAGAAGACGGTGGAGCGCCGCCAACGGCGCATGATCAAGAACCGTGAGTCAGCTGCACGGTCCCGTGCTAGAAAGCAGGCTTATACGGTGGAGCTCGAAGCTGAACTGAACTATCTCAAGCAGGAGAACGCTCGTCTCAAAGAGGCAGAGAAGACGGTTCTACTGACAAAGAAGCAAATGCTGGTTGAGAAAATGATGGAGCAGTCCAAGGAGAAGATGAATGCAAATAGGGGTGGCAGCCAGCTGCGCCGCAGCGGCAGCTGCATGTGGTGA
SEQ ID NO.2：
TAGAAAATTTAATAGAACTTTACAAATTGAGTGTTATAGTGAGATGGCATCGGAGATGAGCAAGAACGTGAAGGTCACCGATGATCAAGAGGTTACATCACAGGAGCGTGACCAAAGTGGTGGTACAAAAGTAGGTGGGGAGGAGGAAATTGCTCCACTGGCGCGGCAGTCGTCAATCCTCTCCCTCACCTTGGAAGAGCTACAAAACTCCTTGTGTGAGCCAGGACGCAACTTTGGTTCCATGAACATGGACGAGTTTGTGGCTAACATATGGAATGCTGAAGAATTCCAGGCTACCACCGGAGGTTGCAAGGGTGCCATGGAGGAAGCCAAGGTGGTAGACAGTGGAAGCGGAAGCGGTGATGCAGGAGGAAGCGGTTTATGTCGGCAGGGATCATTTTCCTTGCCGCTACCGCTGTGCCAGAAGACGGTGGAGGAGGTGTGGACTGAGATCAACCAAGCCCCTGCACACACCTCCGCTCCGGCCTCCGCGCTCCAGCCACATGCCGGGAGCGGTGGTGTTGCAGCTAACGACCGACAGGTAACACTAGGTGAGATGACACTTGAGGATTTCTTGGTAAAGGCCGGGGTGGTCCGAGGGTCCTTTACCGGGCAAGCGGCCATGGGATCTGGCATGGTCAACGGGCCGGTGAACCCCATGCAGCAGGGCCAAGGCGGTCCTATGATGTTCCCAGTAGGACCGGTAAACGCCATGTATCCGGTGATGGGTGATGGCATGGGGTACCCCGGTGGGTACAACGGGATGGCGATTGTGCCACCGCCACCTCCCGCCCAAGGTGCCATGGTTGTCGTGAGTCCTGGATCATCAGATGGGATGAGTGCCATGACACATGCTGATATGATGAATTGTATTGGGAATGGGATGATGATTGAGAATGGAACAAGAAAGCGTCCCCACAGAGAGGATGGCTGCGCCGAGAAGACGGTGGAGCGCCGCCAACGGCGCATGATCAAGAACCGTGAGTCAGCTGCACGGTCCCGTGCTAGAAAGCAGGTACACATGTTGCTCCGAACCTTTTAGTTTCTATGTATCGTTAAGACATTTTGCATGGAGATTGAAATTTTTATGAATAATGCTACAGGCTTATACGGTGGAGCTCGAAGCTGAACTGAACTATCTCAAGCAGGAGAACGCTCGTCTCAAAGAGGCAGAGGTATTAAAGCAAGAAACATTATCCACTCAGCTTTCATTGTCATTCCTAAACAGTCTCGTTCCTTCCTTTTCTGTAACATTGAACTGATAAATATACAATGTCTATATTATCTTCCTTTTACAGAAGACGGTTCTACTGACAAAGAAGCAAATGGTATTGTCCTCTCCCATGTGGCAATCCCCATTCCGCATAGTGCTCATCGTTTTGTGAAATTATTAAATACATATTTGTTATTCATTTATTTGTGTGTGTATATATGCTCATGGAAATATGTCGTGTTCCTGTGATTTTTCAGCTGGTTGAGAAAATGATGGAGCAGTCCAAGGAGAAGATGAATGCAAATAGGGGTGGCAGCCAGCTGCGCCGCAGCGGCAGCTGCATGTGGTGAAACGGGCATAGCGGTGACCGGCGAGTGGCACACTCATCCTGCTTCTGGTGTGGCACTAGCCTCTGAGGCTCTGAAGAGGATTTGCACATTAAGTCTTCTTTCAGACTAAGTGATGTGAAGGTATTATGGTAGTAGTTGATATACATATATGCTGTGATGCTTGTAAACCTGCCTGGACCCACATGTCTTGCCATCGTGTTCACCGGTCCTTGCTGCTTATATGGTTACATGTCGCTGTTTGTACAAAACAACGATGAATAAAATTATACAATAGTATACTAGTTTGTATCTGTAAGTTTGTTCTCATGTCATATGCGCTGGAAAAAGAAAAGCTACAGTATAGGCAAACCTTGGTAGTATCGATTACGGCCCAAATTTCTGGCCGAATAAACTGGAAACCGAAACAAGCTGATGATGATTTGGGCTTTGCGATGGCGAAAA
SEQ ID NO.3：
MASEMSKNVKVTDDQEVTSQERDQSGGTKVGGEEEIAPLARQSSILSLTLEELQNSLCEPGRNFGSMNMDEFVANIWNAEEFQATTGGCKGAMEEAKVVDSGSGSGDAGGSGLCRQGSFSLPLPLCQKTVEEVWTEINQAPAHTSAPASALQPHAGSGGVAANDRQVTLGEMTLEDFLVKAGVVRGSFTGQAAMGSGMVNGPVNPMQQGQGGPMMFPVGPVNAMYPVMGDGMGYPGGYNGMAIVPPPPPAQGAMVVVSPGSSDGMSAMTHADMMNCIGNGMMIENGTRKRPHREDGCAEKTVERRQRRMIKNRESAARSRARKQAYTVELEAELNYLKQENARLKEAEKTVLLTKKQMLVEKMMEQSKEKMNANRGGSQLRRSGSCMW.
the amylose content in the present invention is determined according to the following method:
the detection principle is that amylose and iodine are utilized to form a blue complex: specifically, ethanol is used to separate soluble sugar and starch in the sample, and iodine is used to react with the soluble sugar and starch to obtain the amylose content. The determination was carried out using an amylose content detection kit (BC 4265, solebao) and the specific steps were as follows: weighing 0.01 g of a dried sample, adding 1mL of a first reagent, and fully homogenizing; extracting in water bath at 80deg.C for 30min, centrifuging at 25deg.C for 5 min, discarding supernatant, and collecting precipitate; adding 1mL diethyl ether to the precipitate and oscillating 5 min;3000 g, centrifuging at 25 ℃ for 5 min, discarding the supernatant, and leaving a precipitate; adding 5mL reagent IV into the precipitate for full dissolution, carrying out water bath at 90 ℃ for 10 min, cooling to 3000 g, centrifuging at 25 ℃ for 5 min, and taking the supernatant to be measured. The enzyme label instrument is preheated by more than 30min, and the dual wavelength is regulated to 550nm and 485nm for measurement and calculation.
Example 1 cloning of Rice OsbZIP10 Gene, construction of Gene knockout engineering bacterium and Gene overexpression engineering bacterium
1. Total RNA extraction from rice
Extracting total RNA of young leaves of rice by TIANGEN PLANT total RNA extraction kit, carrying out reverse transcription on the extracted RNA by using a reverse transcription kit of Thermo Fisher company according to instructions to obtain cDNA (shown as SEQ ID NO. 1), and storing at-20 ℃ for later use.
2. Construction of OsbZIP10 gene editing engineering bacterium A
Based on the genomic (SEQ ID NO. 2) sequence, a specific target sequence is searched online (http:// CRISPR. Hzau. Edu. Cn/CRISPR /), the target sequence "GGCACCCTTGCAACCTCCGGTGG" (SEQ ID NO. 4) is selected, and a complementary primer is synthesized according to the target sequence:
OsbZIP10-CRISPR-F：5’- GGCAGCACCCTTGCAACCTCCGG-3’（SEQ ID NO.5）；
OsbZIP10-CRISPR-R：5’-AAACCCGGAGGTTGCAAGGGTGC-3’（SEQ ID NO.6）。
the primers are fused in a PCR instrument to obtain a fusion fragment containing the target sequence.
The vector pHun c12 was digested with BsaI restriction enzymes and the linearized vector was obtained by gel recovery. Subsequently, the fusion fragment was ligated to a linearized pHun c12 vector (Jiang et al ,2019,Mutation of Inositol 1,3,4-trisphosphate 5/6-kinase6 Impairs Plant Growth and Phytic Acid Synthesis in Rice. Plants 8(5): 114）, restriction enzyme identification to obtain the correct vector, and further sequencing to confirm that the target sequence was introduced into the vector, the correct vector was named pHun c12-OsbZIP10. Simultaneously pHun c12-OsbZIP10 was introduced into Agrobacterium strain EHA105 by heat shock transformation, thus obtaining Agrobacterium engineering strain A containing the gene editing vector pHun c12-OsbZIP10 for subsequent genetic transformation.
3. Construction of OsbZIP10 gene over-expression engineering bacterium B
Specific amplification primers of the rice OsbZIP10 gene coding region sequence are designed, and the primer sequences and the enzyme cutting sites carried by the primer sequences are as follows:
OsbZIP10-OE-F：5’-ACTAGGGTCTCGCACCATGGGAGAAGCTAGCAGTAG-3’（SEQ ID NO.7）；
OsbZIP10-OE-R：5’-ACTAGGGTCTCTCGCCGAAGGCTGAATATTGGCTCT-3’（SEQ ID NO.8）。
The nucleotide sequence of the exon of the OsbZIP10 gene is amplified by polymerase chain reaction (Polymerase Chain Reaction, PCR) by using cDNA (Complementary DNA) obtained by reverse transcription of total RNA of leaves of a common rice Japanese plant as a template, and is connected to a carrier pCAMBIA1301 after BsaI digestion and is subjected to thermal shock conversion coating at 42 ℃.
The monoclonal colony is selected, the bacterial liquid after shaking is sent to a sequencing company for sequencing, the sequencing result shows that the vector contains the nucleotide sequence of the exon of the OsbZIP10 gene, the plasmid is extracted by a full-scale gold company plasmid extraction kit, the plasmid is shocked and converted into GV3101 agrobacterium competent, the bacterial liquid is picked up for PCR verification after two days of culture at 28 ℃, and the agrobacterium engineering bacterium B containing the overexpression vector pCAMBIA1301-Ubi is obtained, wherein OsbZIP10 is GFP.
Example 2 obtaining of homozygous plant for OsbZIP10 Gene knockout and homozygous plant for Gene overexpression in Rice
1. Agrobacterium-mediated genetic transformation of rice genes
The agrobacterium engineering bacteria A containing a vector for knocking pHun c12-OsbZIP10 gene and the agrobacterium engineering bacteria B containing pCAMBIA 1301-Ubi::: osbZIP 10::: GFP are used for infecting rice callus, and the corresponding genetically transformed plants are obtained after screening of a differentiation medium and a rooting medium.
2. Obtaining of over-expressed homozygous lines
The T 0 generation over-expressed shoots were taken a few leaves, total RNA was extracted from plant material (root, leaf) using RNEASY PLANT RNA MINI KIT (Qiagen, hilden, germany), and cDNA was reverse transcribed using 1. Mu.g total RNA, oligo-dT18 primer and GoScriptTM reverse transcription system (Promega). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green GoTaq qPCRMaster Mix (Promega, wis., USA). The rice ACTIN gene was used as an internal control and the relative expression level of OsbZIP10 was calculated using the 2 -ΔΔCt method.
TABLE 1 quantitative real-time polymerase chain reaction (qRT-PCR) primer tables
Selfing the T 0 generation transgenic over-expression plants to obtain T 1 generation seeds respectively, taking 6-9 positive plants from the T 1 generation generated by each T 0 generation transgenic over-expression plant, continuing selfing to generate T 2 generation, and carrying out separation analysis. When T 2 generation seedlings generated by the T 1 generation positive strain are all positive strains through detection, the T 1 generation plants are over-expression homozygous strains, namely, transgenic pure lines with over-expression of the OsbZIP10 genes are obtained, and the transgenic pure lines are named as OsbZIP10-ox1 and OsbZIP10-ox2; otherwise, the hybrid strain is obtained.
3. Obtaining of homozygous plants for Gene editing
Young leaves are selected when T 0 generation transgenic rice grows to 3-4 leaf stage, genome DNA is extracted by a CTAB method, pHun c12-OsbZIP10 specific primer (upstream primer: 5'-GCAGGCGGAGCAAGAGCAAC-3'; downstream primer: 5'-GCTGCTCGCTGTCAGGGATG-3') is utilized to amplify endogenous OsbZIP10 genes of the transgenic rice, sequencing and verification of PCR products show that single base deletion of the OsbZIP10 genes is carried out, and the transgenic pure lines with the OsbZIP10 gene function deletion are obtained and named OsbZIP-1 and OsbZIP10-2.
Example 3OsbZIP10 Gene knockout Rice grain direct starch content and related Gene expression determination
OsbZIP10 gene knockout pure line for different rice background materials obtained in example 2: osbZIP10-1 and OsbZIP-2, rice OsbZIP10 overexpresses the T2 generation homozygous lines OsbZIP10-ox1 and OsbZIP10-ox2, and the following analysis was performed in comparison with the ordinary rice material.
1. Rice grain direct-connection starch content determination
The amylose content was determined according to the previous method (Bao et al, 2010). The detection principle is to use amylose and iodine to form blue complex for determination: specifically, ethanol is used to separate soluble sugar and starch in the sample, and iodine is used to react with the soluble sugar and starch to obtain the amylose content. We used the amylose content assay kit (BC 4265, solebao) for the determination, the specific steps are as follows: weighing 0.01 g of a dried sample, adding 1mL of a first reagent, and fully homogenizing; extracting in water bath at 80deg.C for 30 min, centrifuging at 25deg.C for 5min, discarding supernatant, and collecting precipitate; adding 1mL diethyl ether to the precipitate and oscillating 5 min;3000 g, centrifuging at 25 ℃ for 5min, discarding the supernatant, and leaving a precipitate; adding 5mL reagent IV into the precipitate for full dissolution, carrying out water bath at 90 ℃ for 10min, cooling to 3000 g, centrifuging at 25 ℃ for 5min, and taking the supernatant to be measured. The enzyme label instrument is preheated by more than 30 min, and the dual wavelength is regulated to 550 nm and 485nm for measurement and calculation.
The gelatinization properties of the starch, including gum consistency, gelatinization temperature, were measured. The gum consistency of the rice flour was measured using a 3-D viscosity tachometer RVA from company Newport Scientific in australia: when the content is 12.0%, the weight of the rice powder to be added is 3.0g, and distilled water is added to 25.0 mL. The specific heating process is as follows: maintaining 1 min at 50deg.C; raising to 95 deg.c at constant speed (3.8 min); maintaining at 95deg.C for 2.5 min; then the temperature was lowered to 50℃at a constant rate (3.8 min), and maintained at 50℃at 12.5: 12.5 min. The stirrer was rotated at an initial 10 s rotation rate of 960 r/min, after which it was maintained at 160 r/min. RVA profile is characterized by the highest viscosity, hot slurry viscosity and final viscosity. The gelatinization temperature of the rice flour was measured using a differential scanning calorimeter (DSC 2920 Modulated DSC thermal analyser,TA Instruments,Newcastle,DE): the rice flour 2.0 mg was weighed into an assay aluminium pot, 6L double distilled water was added, the aluminium pot was sealed with a lid, equilibrated at room temperature for 1h, and then heated from 30℃to 110℃at a rate of 10℃per minute. An empty can was used as a reference. Parameters such as the gelatinization initial temperature, the highest temperature, the final temperature and the like are obtained by analysis in matched general analysis software.
The results are shown in fig. 1 and 2, and the OsbZIP10 gene knockout pure line of different rice background materials: osbzip10-1 and osbzip-2, the amylose content is significantly lower than that of its wild-type material, and the consistency and gelatinization temperature are significantly higher than that of its wild-type material; the rice OsbZIP10 over-expresses T2 generation homozygous lines, namely OsbZIP10-ox1 and OsbZIP10-ox2, the amylose content is obviously higher than that of wild type materials, and the gum consistency and gelatinization temperature are obviously lower than those of the wild type materials.
2. Expression level measurement of sucrose Metabolic Gene (OsBGLU), lipid Metabolic Gene (OsLTP 2 and OsOLE) and active oxygen scavenging Gene (OsHSFC 1B)
The above-mentioned snapping material was taken seven days after flowering of rice, total RNA was extracted from the material using RNEASY PLANT RNA MINI KIT (Qiagen, hilden, germany), and cDNA was reverse transcribed using 1. Mu.g total RNA, oligo-dT18 primer and GoScriptTM reverse transcription system (Promega). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green GoTaq qPCRMaster Mix (Promega, wis., USA). The rice ACTIN gene was used as an internal control and the relative expression levels of OsBGLU, osLTP2, osOLE18 and OsHSFC1B were calculated using the 2 -ΔΔCt method.
As shown in FIG. 3, the expression levels of sucrose metabolic gene (OsBGLU), lipid metabolic gene (OsLTP and OsOLE) and scavenger active oxygen gene (OsHSFC B) were shown in the OsbZIP10 gene knockout pure line of different rice background materials: osbzip10-1 and osbzip10-2 are significantly lower than their wild-type materials; the rice OsbZIP10 over-expresses T2 generation homozygous lines, namely OsbZIP10-ox1 and OsbZIP10-ox2, which are significantly higher than wild type materials thereof. This suggests that the OsbZIP10 gene may affect amylose content by balancing carbon partitioning and ROS homeostasis in rice kernels.
Claims (9)
- The application of the OsbZIP10 gene in regulating and controlling the amylose content of rice grains is characterized in that the nucleotide sequence of a protein coding region of the OsbZIP10 gene is shown as SEQ ID NO. 1; the regulation and control method comprises the following steps: (1) The OsbZIP10 gene is knocked out, so that the amylose content in rice grains is reduced; or (2) through over-expressing the OsbZIP10 gene, the amylose content in the rice grains is improved.
- 2. The application of the bZIP family transcription factor coded by the rice OsbZIP10 gene in regulation and control of rice grain amylose is characterized in that the amino acid sequence of the bZIP family transcription factor is shown as SEQ ID NO. 3; the regulation and control method comprises the following steps: (1) The OsbZIP10 gene is knocked out, so that the amylose content in rice grains is reduced; or (2) through over-expressing the OsbZIP10 gene, the amylose content in the rice grains is improved.
- 3. The use according to claim 2, wherein in method (2), the bZIP family transcription factor increases amylose content of rice kernels by enhancing sucrose metabolism, lipid metabolism and ability to scavenge active oxygen in rice kernels.
- 4. The application of the related biological material of the rice gene OsbZIP10 in regulating and controlling the amylose content of rice grains is characterized in that the related biological material comprises the following components: an over-expression vector or a genetically engineered bacterium, a CRISPR/Cas9 vector or a genetically engineered bacterium for knocking out the OsbZIP10 gene; the regulation and control method comprises the following steps: (1) The OsbZIP10 gene is knocked out, so that the amylose content in rice grains is reduced; or (2) through over-expressing the OsbZIP10 gene, the amylose content in the rice grains is improved; the nucleotide sequence of the protein coding region of the OsbZIP10 gene is shown as SEQ ID NO. 1.
- 5. A method for regulating amylose content in rice kernels, comprising:(1) The OsbZIP10 gene is knocked out, so that the amylose content in rice grains is reduced;Or (2) through over-expressing the OsbZIP10 gene, the amylose content in the rice grains is improved;the nucleotide sequence of the protein coding region of the OsbZIP10 gene is shown as SEQ ID NO. 1.
- 6. The method according to claim 5, wherein: the method for knocking out the OsbZIP10 gene comprises the following steps:(1) According to the OsbZIP10 genome sequence, designing target sequence sgRNA, and constructing a CRISPR/Cas9 vector edited by the OsbZIP10 gene;(2) Transferring the CRISPR/Cas9 vector into an agrobacterium competent cell, and constructing a genetic engineering bacterium containing the CRISPR/Cas9 vector of the knocked-out OsbZIP10 gene;(3) Culturing the agrobacterium engineering bacteria to mediate transformation of rice callus to obtain a homozygous strain knocking out the OsbZIP10 gene.
- 7. The method according to claim 6, wherein the nucleotide sequence of the target sequence sgRNA is shown in SEQ ID No. 4.
- 8. The method of claim 6, wherein in the preparation of the CRISPR/Cas9 vector of step (1), the upstream primer is shown as SEQ ID No.5 and the downstream primer is shown as SEQ ID No. 6.
- 9. The method of claim 5, wherein the method of overexpressing OsbZIP10 gene is:(1) Designing a primer sequence by taking wild rice cDNA as a template, and constructing an overexpression vector of the OsbZIP10 gene;(2) Transferring the vector into an agrobacterium competent cell to obtain agrobacterium for over-expressing the OsbZIP10 gene;(3) Culturing the agrobacterium engineering bacteria to mediate transformation of rice callus to obtain a homozygous strain of the overexpression OsbZIP10 gene.
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