Rice GDSL lipase coding gene OsHGZ1 mutant and application thereof

Abstract

The invention discloses a mutant of a rice GDSL lipase gene OsHGZ1 and application of the mutant in increasing the zinc content in grains. According to the present invention, the mutant with the mutation of the 195-site amino acid of the OsHGZ1 is obtained through EMS mutagenesis, and the verification results show that the zinc content of the mutant grain is twice the zinc content of the wild type; the OsHGZ1 is an excellent gene which can be applied to cultivation of high-zinc rice grains. The invention has very important significance for improving the nutritional quality of rice and improving the dietary health of human beings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and relates to a rice GDSL lipase gene OsHGZ1 mutant and application thereof in regulating the zinc content in grains. Background Art

[0002] Zinc (Zn) is an essential trace nutrient for plants and animals. It is mainly found in seafood and animal offal, and is present in very small amounts in other foods. It plays a structural and catalytic role in many proteins and participates in a variety of physiological and biochemical processes. The content of zinc in the human body is extremely low, about 2g, but it plays a very important role in human life and health. Since zinc-deficient soils are prevalent worldwide, it is a limiting factor for crop production on millions of hectares of arable land, especially in alkaline soils, so zinc deficiency in crops and humans is a serious problem that affects crop productivity and human health.

[0003] Rice is the staple food of half of the world's population and the main source of dietary zinc for people who rely on rice, but rice cannot provide enough Zn for the human body. There are two reasons for this: one is that 50% of rice field soils are zinc deficient; the other is that 40% of the Zn in brown rice is lost during rice processing. In countries where rice is the staple food, the average annual per capita rice consumption is 62 to 190 kg, which means that trace element malnutrition is very common in developing and underdeveloped countries where cereals are the staple food. Therefore, increasing the zinc content of rice is very important for improving human health.

[0004] Breeding new varieties rich in zinc is an effective way to increase the zinc content of rice. However, since the zinc content of rice is not only controlled by genetics, but also affected by environmental factors, the progress of rice zinc nutrition breeding has been slow. Li Zhigang and others found that the zinc content in rice increases with the increase of zinc content in the environment, but when the zinc supply in the environment reaches a certain level, the increase in the zinc content of rice is not obvious. Zheng Xiaolin and others believe that the ability to transport and distribute zinc to grains is the key to affecting the zinc content of grains. The Rice Research Institute of Anhui Academy of Agricultural Sciences screened a zinc-rich germplasm from the rice germplasm introduced by IRRI, namely IR68144-213-2-2-3 (S5), whose zinc content reached 352.61 mg / kg. Song Wenchang found that the zinc content in black rice is as high as 109.7 mg / kg, which is 2 to 4 times higher than that of ordinary rice.

[0005] GDSL esterase / lipase proteins are hydrolases widely distributed in plants, animals and microorganisms. Research on the plant GDSL lipase family started relatively late, but due to its special structure and multifunctional characteristics, plant GDSL lipases have gradually gained attention and research. More and more studies have shown that GDSL enzymes play an important role in many physiological processes of plants, such as oil seed metabolism, stress, response to plant hormones, and morphological construction of the cuticle or other organs.

[0006] However, current research on the rice OsHGZ1 gene found that complete knockout of the OsHGZ1 gene rendered the plants sterile, and there is still a lack of research on the regulation of rice traits by the GDSL lipase gene. Summary of the invention

[0007] A rice GDSL lipase encoding gene mutant, wherein the 584th C in the CDS sequence region of the wild-type rice OsHGZ1 gene is mutated to T, resulting in the 195th amino acid encoded by it being changed from serine to phenylalanine; the wild-type rice OsHGZ1 gene CDS sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the rice GDSL lipase encoding gene mutant is shown in SEQ ID NO.2.

[0009] The second object of the present invention is to provide the use of the aforementioned rice GDSL lipase encoding gene mutant in increasing the zinc content in rice grains and / or maintaining the rice's ability to propagate.

[0010] The third object of the present invention is to provide a method for screening rice germplasm with high zinc accumulation, comprising the following steps:

[0011] (1) A mutant pool was obtained through EMS chemical mutagenesis, and stable germplasm was obtained after three generations of breeding;

[0012] (2) Hulling and digesting the rice in the mutant library, and determining the element content using inductively coupled plasma mass spectrometry (ICP-MS);

[0013] (3) Obtain rice germplasm resources for producing high-zinc rice based on the zinc content.

[0014] The beneficial effects of the present invention are:

[0015] Provided is an excellent gene OsHGZ1 gene that can be used to cultivate high-zinc rice grains. A mutant in which the amino acid at position 195 of the OsHGZ1 is mutated is obtained by EMS mutagenesis, and it is verified that the zinc content of the mutant grain is twice that of the wild type. At the same time, the present invention overcomes the problem of plant sterility after the complete knockout of the OsHGZ1 gene, the mutant can be propagated in the field, and the high-zinc phenotype can be stably inherited and is not affected by the region and the generation. The present invention is of great theoretical and practical significance for improving the nutritional quality of rice and improving human dietary health. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the grain zinc content in the rice GDSL lipase gene OsHGZ1 mutant and wild-type rice in Example 1;

[0017] Figure 2 To implement 2 results of zinc content in rice GDSL lipase gene OsHGZ1 mutant and wild type rice during grain filling;

[0018] Figure 3 The gene structure and mutation location of OsHGZ1 in Example 1;

[0019] Figure 4 The results of the lipase activity of rice anthers of the rice GDSL lipase gene OsHGZ1 mutant and wild-type rice in Example 3;

[0020] Figure 5 The complemented mutant (COM) obtained in Example 2, the rice GDSL lipase gene OsHGZ1 mutant obtained in Example 1, and the thousand-grain weight of wild-type rice grown in the field;

[0021] Figure 6 The zinc content in the grains of the complemented mutant (COM) obtained in Example 2, the rice GDSL lipase gene OsHGZ1 mutant (hgz1) obtained in Example 1, and wild-type rice. DETAILED DESCRIPTION

[0022] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0023] Example 1 Obtaining a Rice GDSL Lipase Gene OsHGZ1 Mutant

[0024] (1) Construction of rice ethyl methanesulfonate (EMS) chemical mutagenesis library: About 1 kg of rice seeds (Zhonghua 11) were washed with tap water and soaked at room temperature for 20 h. After soaking, the seeds were washed several times with tap water. After draining the water, 0.8% (mass / volume concentration) EMS was added to immerse all the seeds in the fume hood and mutagenesis was carried out at room temperature for 12 h. After the mutagenesis, the seeds were washed twice with tap water in the fume hood, and 3M NaOH was added to the waste liquid and EMS mutagenesis solution to a final concentration of 1 M, and the waste liquid was poured into a bucket. After washing twice, the seeds were washed three times in a sink, and the seeds with more seeds were placed in a sink and rinsed with running water for 3 h. The EMS-mutated rice seeds were sown in the Nanjing base, and moved to the rice field after two weeks of seedling cultivation, with one plant per hole. Three months later, the M1 generation seeds, i.e., the M2 generation, were harvested. The M2 generation was propagated at the base to obtain the M3 generation, which was dried, threshed and stored to complete the construction of the rice mutant library.

[0025] (2) Screening of rice mutants and gene cloning: Select 6 plump seeds with uniform growth from the obtained M3 generation EMS-induced seeds, remove the husks, put them in a centrifuge tube and mark them for later use. Weigh the seeds with the husks removed and record the grain mass. Transfer the weighed grains to a digestion tube, add 1 ml of concentrated nitric acid to each tube, and digest them in a graphite digestion furnace. In the third stage, when the digestion tube is clear and there is no obvious solid matter, only a drop of oily liquid remains, take out the digestion tube, cool it, add 10 ml of 2% nitric acid to make up the volume, centrifuge and take 2 ml of supernatant and transfer it to a 2 ml centrifuge tube, and use inductively coupled plasma mass spectrometry (ICP-MS) to determine the element content.

[0026] (3) The mineral element content data of 3200 rice EMS-induced mutagenesis seeds were compared with the zinc content. The 1% of rice plants with the highest zinc content in the grain were recorded and selected. Subsequently, the seeds were bred in two locations for three years (in Lingshui, Hainan in the winter and in Nanjing in the summer) to verify the stability of the grain zinc content phenotype. Finally, the rice GDSL lipase gene OsHGZ1 mutant (hgz1) was obtained.

[0027] The experimental results are as follows Figure 1As shown: 6 mutants (hgz1) and wild types were planted in the field, and the middle 4 strains were selected for sampling. 6 grains were taken from each strain and dried to constant weight, then shelled and digested. The digestion solution was diluted with 2% nitric acid and the mineral element content of the samples was determined using ICP-MS. The results showed that the zinc content of the mutant (hgz1) grains was twice that of the wild type, and the difference was extremely significant. The above results indicate that the mutant can reproduce normally in the field, and the high zinc phenotype can be stably inherited and is not affected by region and generation. The target gene OsHGZ1 (LOC_Os02g18870) was determined by molecular biological means. Since the gene has been reported, the gene positioning process will not be repeated here. The gene structure and mutation location of OsHGZ1 are shown in Figure 2. Figure 3 As shown: OsHGZ1 gene has 5 exons and 4 introns. The mutation protected by this patent occurs in the third exon, and the 195th amino acid changes from serine to phenylalanine, resulting in an increase in the zinc content of the mutant grains.

[0028] Example 2 Obtaining a rice GDSL lipase gene OsHGZ1 complemented mutant

[0029] In order to confirm that LOC_Os02g18870 is the gene HGZ1 that controls high zinc in grains, the entire genome of LOC_Os02g18870 and the promoter and termination regions (6.5 kb in total) were connected to the complementary vector pCAMBIA1301 between the BamHI and HindⅢ restriction sites and transformed into the callus of the mutant (hgz1) identified by PCR. A total of 10 independent rice transgenic positive plants hgz1COM (COM) were obtained by PCR and electrophoresis detection. Figure 6 The transgenic plants (COM) shown here restored the wild-type grain zinc content and the thousand-grain weight to normal ( Figure 5 ), which confirmed that LOC_Os02g18870 is the gene HGZ1 controlling high zinc in grains.

[0030] Example 3 Determination of zinc content in grains of mutants during the grain filling period

[0031] The seeds of the mutant (hgz1) and the wild type of Zhonghua11 were marked 7, 23, and 32 days after flowering, and the filling time of different batches was recorded. Four biological replicates were set for each sample during sampling. Due to the small biomass, 10 seeds were collected for each replicate at 7 days, and 6 seeds were collected at 23 days and 32 days. The samples were digested by graphite digestion and the element content was determined by ICP-MS.

[0032] The experimental results are as follows Figure 2The results show that the zinc content in the grains of the mutant (hgz1) was significantly higher than that of the wild type throughout the filling period, and the change in zinc content in the early filling period (0d-7d) was the largest (the increase was 95%), and the zinc content in the middle and late filling period (7d-23d) maintained an increasing trend but the amplitude decreased. The above results show that the zinc content in the grains of the hgz1 mutant mainly accumulated in large quantities in the early filling period, and continued to increase in the late filling period but the increase was small.

[0033] Example 4 Determination of mutant lipase protease activity

[0034] (1) Plant total protein extraction Using the Plant Protein Extraction Kit, rice panicles of the mutant (hgz1) and wild type (WT) were taken, and three biological replicates were set up for subsequent lipase activity determination. During the flowering period, anthers were taken during the flat leaf pulvinus period, that is, the pollen mother cells were in the meiotic period. In the field, 3-5 panicles were selected according to the growth morphology of the plants and then sent to the laboratory for subsequent experiments. The lipase activity experiment used a lipase (LPS) activity kit, which uses p-nitrophenol ester as a substrate. Lipase hydrolyzes the substrate to produce colored p-nitrophenol. The absorbance value is measured at a wavelength of 405nm to obtain the lipase activity. The protein concentration of the total plant protein obtained by measuring the BCA protein concentration kit was diluted according to the result.

[0035] (2) Prepare a standard curve; prepare a standard stock solution (20 μmol / mL): add 1 ml of distilled water to the standard EP tube, dilute the stock solution into the following concentration gradient standards: 0, 0.4, 0.8, 1.2, 1.6, 2 μmol / mL, read the absorbance at 405 nm, and prepare a standard curve. Enzyme activity determination: preheat the microplate reader for 30 minutes, adjust the wavelength to 405 nm, and adjust the value to zero with distilled water. Use a cuvette to measure the light absorption value at a wavelength of 405 nm, starting from 0 min and measuring every 5 min for a total of 60 min, and record the light absorption value of each reaction for comparison. In this example, p-nitrophenylbutyrate was used as a substrate to detect the lipid hydrolase activity of the HGZ1 protein, and the total protein extracted from the wild-type anthers of Zhonghua 11 and hgz1 was used to detect the lipase activity, and a sample without plant total protein was used as a negative control.

[0036] The experimental results are as follows Figure 4 As shown: The hydrolase activity of the mutant was significantly reduced compared to the wild type, indicating that the level of enzyme activity of hgz1 was reduced due to the amino acid variation.

[0037] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rice GDSL lipase encoding gene mutant, characterized in that: The rice GDSL lipase encoding gene mutant is a wild-type rice OsHGZ1 gene CDS sequence region in which the C at position 584 is mutated to T, resulting in the conversion of the encoded amino acid at position 195 from serine to phenylalanine; the wild-type rice OsHGZ1 gene CDS sequence is shown in SEQ ID NO.

1.

2. The rice GDSL lipase encoding gene mutant according to claim 1, characterized in that: The amino acid sequence of the rice GDSL lipase encoding gene mutant is shown in SEQ ID NO.

2.

3. Use of the rice GDSL lipase encoding gene mutant according to claim 1 in increasing the zinc content in rice grains and / or maintaining the ability of rice to be propagated.

4. Claim 1 comprises a method for screening rice germplasm with high zinc accumulation, characterized in that: The steps include: (1) A mutant pool was obtained through EMS chemical mutagenesis, and stable germplasm was obtained after three generations of breeding; (2) The rice in the mutant library was hulled and digested, and the mutants were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Plasma mass spectrometry, ICP-MS) was used to determine the element content; (3) Obtain rice germplasm resources for producing high-zinc rice based on the zinc content.

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