CN110964735B - Application of rice gene OsHXK9 in regulation and control of seed dormancy - Google Patents

Abstract

The invention discloses an application of a rice gene OsHXK9 in regulation and control of seed dormancy, wherein a genomic DNA sequence of the rice gene OsHXK9 is shown as SEQ ID No. 1. The rice mutant OsHXK9 with OsHXK9 gene function deletion is obtained by using a gene editing technology, the sprouting of the mutant ear is obviously delayed, and the mutant has very important application in agricultural production; meanwhile, the invention provides an efficient breeding method for new rice varieties, germplasm resources and hybrid rice parents with obviously delayed sprouting of the panicle based on the characteristics of OsHXK9 gene and seed sprouting.

Description

Application of rice gene OsHXK9 in regulation and control of seed dormancy

Technical Field

The invention relates to the technical field of plant genetic engineering and rice molecular breeding, in particular to application of a rice gene OsHXK9 in regulation and control of seed dormancy.

Background

Seed dormancy is an adaptation of plants to the formation of adverse environments in the long-term phylogenetic process and is also an important physiological stage in the life activities of plants. For agricultural production, seed dormancy is a double-edged sword.

On the one hand, a reduced or even lost dormancy of seeds will adversely affect timely harvest of the crop (Shu et al, Do fashion and cultivation: How the crop seed settled plant Biol 2015,17:1104 1112; Shu et al, Two faces of one seed: thermal regulation of farmland and cultivation on. mol plant 2016,9: 34-45). For example, in rice production, due to abnormal global climate change, disaster weather frequently occurs, and the occurrence of phenomena of pre-harvest sprouting and large-area 'sprout' of rice is often caused by the fact that the late growth period of rice encounters rainy and poor-exposure weather more frequently than before. In addition, in recent years, hybrid rice varieties widely popularized and planted in China are generally short or unobvious in the dormancy stage and serious in the sprouting phenomenon of ears. In south, especially in the middle and lower reaches of Yangtze river, about one third of the year is in rainy days in the rice mature period, the problem of pre-harvest sprouting is particularly obvious, and the problem becomes a general problem to be solved urgently in the production of late rice (including single-season late rice and double-season late rice) in south of China. For example, nearly 7 ten thousand mu of rice (including Zhengnuo 19, Nanjing 9108, nephrite, Nanjing 51, Wuyujing 23, Wuyujing 30 and other rice varieties) has the phenomenon of sprouting (http:// www.cnjidan.com/n ews/888885) in Jiangsu Yangxi, which seriously affects the harvest yield and rice quality of the rice in 2016. In addition, in the process of producing and propagating hybrid rice in south China, due to the increase of the application amount of '920' (gibberellin), hybrids, particularly hybrid seed production of the series such as gang you, D you, II you and super you are often threatened by ear sprouting, the ear sprouting rate in normal years is 5-10%, the ear sprouting rate in special years can reach 20-30%, and in severe cases can reach 60-80% (Xubaochi, research on genetic improvement and utilization of dormancy of hybrid indica parent seeds, 2009, doctor thesis, Nanjing university of agriculture), so that the utilization value of the hybrids is greatly influenced, and the worldwide problem is urgently solved in hybrid seed production for decades.

On the other hand, the Dormancy of the seeds is too strong, which can cause the seeds not to germinate or the emergence of the seeds is uneven, which affects the production process of the rice and is difficult to adapt to the requirements of the modern agriculture on scale and standardized production (Shu et al, society and age conservation: How the crop seed plant Biol 2015 17:1104 + 1112; Shu et al, T wo faces of one seed: geographical regulation of history and management. mol plant 2016,9: 34-45). However, the high yield, high quality, disease and pest resistance and other indexes of rice breeding are considered more in many years, so that the preservation of proper dormancy of seeds is ignored, and in addition, in recent years, the increase of rainy and short-time weather at the late stage of rice production causes the defect that many high-yield hybrid rice and conventional rice are easy to spike and sprout.

Therefore, the molecular physiological mechanism of rice seed dormancy is deeply researched, the rice variety with proper dormancy is cultivated, various problems caused by the rice dormancy characteristic are fundamentally solved, the rice production loss is reduced, the production cost is reduced, and the method has very important significance for guaranteeing the food safety in China.

Regarding the cause of seed dormancy, a great deal of research has been carried out at home and abroad from the perspective of the morphological structure of seeds and the molecular physiology of dormancy regulation and release for many years, and a number of important academic points or theoretical hypotheses have been proposed so far, including the theoretical hypotheses such as endogenous hormone regulation theory (the three-factor theory), respiration pathway theory (pentose phosphate pathway), phytochrome regulation theory, membrane phase change theory and cytoplasmic pH (Xubaokui, research on genetic improvement and utilization of dormancy of hybrid indica parent seeds 2009, doctor, Nanjing university of agriculture; Shengzhong loyalty, etc., research progress on rice seed dormancy QTL localization and molecular mechanism, crop research, 2009, 23: 318-.

Although these hypotheses explain to some extent the physiological regulation problem of seed dormancy, they are not sufficient to truly elucidate the mechanism of seed dormancy. Among them, one of the important deficiencies of the current hypothesis and the previous related research work is: the endogenous hormone regulation theory over-emphasizes the action of the relevant hormones of seed dormancy, such as gibberellin, abscisic acid, auxin and the like; the respiratory pathway theory emphasizes the important role of oxygen in seed dormancy breaking. However, the dormancy of seeds is not completely determined by a certain aspect or a certain simple physiological factor, and a plurality of physiological metabolic changes and the fine regulation of related genes are involved in the process of dormancy and dormancy breaking. Thus, the underlying mechanism for inducing, delivering, maintaining and completing seed dormancy has not been clear so far, and the dormancy mechanism is also a "gray box".

In the case of rice, the cause of seed dormancy is various, and is not controlled by a single factor, but is probably caused by multiple factors, and the factors influence each other.

First, the ABA/GA ratio is one of the important physiological factors affecting rice Seed dormancy (Liu et al, sequence and expression analysis of Seed-specific and expression-associated AB A-and GA-related genes in rice Seed front Plant Sci.2011,2: 17; Liu et al, expression patterns of ABA and GAmeta-genes and hormone expression vector Seed dependent dev. expression and inhibition: a composition of rice Seed and non-rice Seed genes J Gene applications.2014: 327-38; Shu et al, Two aspects of rice Seed one of nutritional and physiological regulation of rice Seed mol.2016: 2016.9: 2016). Studies have shown that the ABA/GA ratio is positively correlated with seed dormancy (Liu et al, Sequence variation and Expression analysis of seed correlation-associated ABA-and GA-related genes in rice significance, front nt Plant Sci.2011,2: 17; Liu et al, Expression patterns of ABA and GA correlation generation and hormone level reduction rice significance: a comparison of d-order and non-point rice significance. J Gene significance. 2014,41: 327-38).

Secondly, the root cause of seed dormancy is determined by genotype or gene. Wild rice is the ancestor of cultivated rice, germplasm resources are rich, the function of a dormant gene is not lost in the evolution process, and some varieties retain or lose the characteristic in the domestication process of the cultivated rice. In recent years, some scholars have initially positioned dormancy genes for controlling rice seeds by using methods such as QTL (quantitative trait locus) and the like, and the results show that the number of QTL related to rice dormancy is about 140, and the QTL related to rice dormancy is mainly distributed on No.1, No. 3, No. 5, No. 6, No. 7 and No. 11 chromosomes of rice, but the QTL sites which are finely positioned or cloned are very few (Haoming, QTL analysis of rice variety 4628 dormancy and construction of dormancy near isogenic lines. 2014, Master thesis, Hunan agriculture university). In addition, the dormancy characteristics of rice seeds are often not controlled by a single major gene, but rather as a result of the co-action of multiple major or minor genes. ToRice genes which have been cloned in connection with seed dormancy are Serine carboxypeptidase 46(Li et al, rice carbohydrate 46 regulation genes profiling and seed promotion in rice (Oryza sativa L.). 2016,11: e0159737), GA synthetic gene OsGA20ox2(Ye et al, Map-based regulation of seed stimulation 1-2 identified a rice gene regulation of seed stimulation gene. plant physiology.2015, 169:2152-2165), ABA receptor gene OsPYL/AR 5(K im et al, A nucleotide of protein synthesis, P/P gene expression, P/P gene of rice protein synthesis, protein synthesis of rice protein, expression of rice protein, growth promoter, protein synthesis of rice protein, expression of rice protein, rice protein synthesis of rice protein, rice protein synthesis of rice protein, rice protein synthesis of rice protein, rice protein synthesis of rice protein, rice protein synthesis of rice, 54:177-⁺The ATP ase gene VHA-A1(Yang et al, A single cysteine deletion in the OsPLS1 gene encoding vacuolar-type H⁺-ATPase subunit A1 leads to premature leaf senescence and seed dor mancy in rice.J Exp Bot.2016,67:2761-2776)。

Disclosure of Invention

The invention provides a new application of a rice gene OsHXK9 in regulating and controlling seed dormancy, and the biological function of the gene can delay the germination time of rice ears after being reduced or destroyed.

The specific technical scheme is as follows:

the invention provides application of a rice gene OsHXK9 in regulation and control of seed dormancy, wherein a genome sequence of the rice gene OsHXK9 is shown as SEQ ID No. 1.

When the function of the rice myoglucose kinase gene OsHXK9 is researched, the fact that OsHXK9 is used as a positive regulation and control factor of seed germination in normal rice can promote seed germination is found, after the gene is knocked out by using a gene editing technology, the fact that the agronomic characters of mutant offspring are basically not influenced is found, but the sprouting of ears of mutant offspring is obviously delayed, and therefore the gene has the capability of regulating and controlling rice seed dormancy.

Specifically, the regulation and control approach is to reduce or destroy the biological function of the OsHXK9 gene in the rice genome and delay the germination of the rice ears.

Specifically, the application of the rice gene OsHXK9 in regulation and control of seed dormancy comprises the following steps:

(1) constructing a recombinant expression vector for editing, interfering or knocking out the OsHXK9 gene by using a gene editing technology;

(2) and transferring the recombinant expression vector into a host cell, and then transforming a receptor plant to obtain a strain with reduced or damaged biological function of the OsHXK9 gene.

Wherein, the receptor plant can be rice or arabidopsis thaliana, and preferably, the receptor plant is rice.

Further, the host cell is agrobacterium.

Further, the agrobacterium is agrobacterium EHA 105.

Compared with the prior art, the invention has the following beneficial effects:

the mutant rice strain OsHXK9 with OsHXK9 gene function deletion is obtained by using a gene editing technology, the sprouting of the ear of the strain is obviously delayed, and the mutant rice strain has very important application in agricultural production; meanwhile, the invention provides an efficient breeding method for new rice varieties, germplasm resources and hybrid rice parents with obviously delayed sprouting of the panicle based on the characteristics of OsHXK9 gene and seed sprouting.

Drawings

FIG. 1 shows the results of sequencing analysis of oshxk9 mutation sites in wild type rice and mutant rice in example 2.

FIG. 2 shows the sprouting results of the wild type and its mutant oshxk9 in example 3;

wherein A is the phenotype of the 4 th day of ear germination; and B is the result of the dynamic analysis of the germination rate of the ear.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

The following examples used molecular biological and biochemical methodsA Laboratory Manual, 3, Molecular Cloning, published by Cold Spring Harbor Laboratory Press (2001), written by Ausubel, Current Protocols in Molecular Biology and J.Sambrook et al, published by John Wiley and Sons, Inc^rdED., etc., are described in detail. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

Example 1OsHXK9 Gene knockout vector construction

Based on the genome (SEQ ID NO.1) sequence, a specific target sequence was found online (http:// crispr. dbcls. jp /), a target sequence "GCACCAGAGGCAACGTCGGG" was selected, and complementary primers were synthesized from the target sequence:

an upstream primer: 5'-GGCAGCACCAGAGGCAACGTCGGG-3', respectively;

a downstream primer: 5'-AAACCCCGACGTTGCCTCTGGTGC-3', respectively;

and fusing the primers in a PCR instrument to obtain a fused fragment containing a target sequence.

The vector pHun4c12 was digested with BsaI restriction enzyme, and the gel was recovered to obtain a linearized vector. The above fusion fragment was ligated into linearized pHun4c12(Xu et al 2014, Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice 7:5), and the correct vector obtained by the enzyme digestion identification and further sequencing was confirmed to have the target sequence introduced into the vector, which was named pHXK 9. At the same time, pHXK9 was introduced into Agrobacterium strain EHA105 by electrical transformation for subsequent genetic transformation.

Example 2 obtaining of OsHXK9 Gene knockout transgenic pure line OsHXK9

Step 1 genetic transformation of transgenic Rice

Genetic transformation of rice was performed according to the method of Pan et al (2006) with minor modifications.

Briefly described as follows: inoculating sterilized seed B of QIANJIANG No. 6 to seed containing 2.5mg L ^-12,4-D of N₆Culture medium, inducing callus under 32 deg.C continuous illumination for 7-10 days; about 40. mu.L of the EHA105 Agrobacterium solution containing the pHXK9 plasmid was added to 25mg L^-1Rif and 50mg L^-1Kan's 20mL LB liquid medium, 28 ℃ overnight shake cultureThe cells were collected by centrifugation and 10mM MgSO₄Resuspending the bacterial suspension and centrifuging, removing supernatant, and resuspending to bacterial suspension OD with AA liquid medium containing 200 μ M AS₆₀₀0.1-0.2; infecting the callus in bacterial liquid for 30min, transferring the callus to sterilizing filter paper, and sucking off excessive bacterial liquid; transferring the callus to a co-culture medium on a CC-AS culture medium, performing dark culture at 28 ℃ for 55-60 h, and transferring the callus to a medium containing 250mg L^-1N of Cef₆A culture medium; dark culture at 28 deg.C for 7d, transferring the callus to 250mg L^-1Cef and 50mg L^-1N of Hyg₆Continuously screening and culturing on the culture medium to obtain resistance callus; the resistant callus is regenerated and cultured to obtain T₀Transgenic seedlings are planted in the field until T is harvested₁。

Step 2 molecular identification of transgenic rice

Will T₀Young leaves are selected when the generation transgenic rice grows to 3-4 leaf stage, genome DNA is extracted by a CTAB method, and a specific primer HXK9 (an upstream primer: 5'-CAATTACTGCTGCTTTGACC-3'; a downstream primer:

5'-AAGAAGTCCCTCTCTTCGAT-3') amplifying endogenous OsHXK9 gene of the transgenic rice, and sequencing and verifying PCR products, wherein the result shows that the OsHXK9 gene has single base deletion (figure 1), thus obtaining a transgenic pure line with OsHXK9 gene function deletion, which is named OsHXK 9.

Example 3 comparative analysis of ear sprouting in OsHXK9 Gene knock-out transgenic inbred OsHXK9

Wild-type rice qianjiang No. 6B and oshxk9 were planted in the field, normal fertilizer and water management, and after the rice was mature, 20 individual plants were randomly selected for seed test, and the results showed that there was no significant difference between the two (table 1).

TABLE 1 major agronomic traits of mutants and their wild-type

Then, the mature rice ears are placed in a culture dish to carry out germination tests under the condition of 30 ℃, and the ear germination rate is counted every 1 day. The results showed that the ear germination of oshxk9 was significantly delayed (fig. 2), with the ear germination rate reaching 98.24% on day 4 of the wild type, whereas the mutant did not substantially germinate at this time (fig. 2A), and the mutant did not germinate until 85.46% on day 9, beginning on day 5.

Sequence listing

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Claims (4)

1. Rice geneOsHXK9Application in regulation of dormancy of rice seeds, which is characterized in that the rice baseDue to the fact thatOsHXK9The gene sequence of (A) is shown in SEQ ID NO. 1;

the regulation is realized by reducing or destroying the rice genomeOsHXK9The biological function of the gene delays the rice ear germination.

2. The use according to claim 1, comprising the steps of:

(1) construction of interferents or knockouts using gene editing techniquesOsHXK9Recombinant expression vectors for the genes;

(2) transferring the recombinant expression vector into host cells, and transforming a receptor plant to obtain the recombinant expression vectorOsHXK9A strain in which the biological function of a gene is reduced or disrupted.

3. The use of claim 2, wherein the host cell is agrobacterium.

4. The use of claim 3, wherein the Agrobacterium is Agrobacterium EHA 105.

Images