CN111793633B - Application of OsNRT2.3b in improving yield and rice quality - Google Patents
Application of OsNRT2.3b in improving yield and rice quality Download PDFInfo
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Abstract
The invention discloses application of OsNRT2.3b in improving yield and rice quality. Under different fertilizer treatment conditions, the effective tillering, single ear weight, single plant yield, brown rice rate and polished rice rate of the total OsNRT2.3b super-expression material are higher than those of a Nipponbare wild type; the moisture content and aspect ratio are higher than those of the Japanese clear type, and the chalky grain rate and chalky degree are both lower than those of the Japanese clear type.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to application of OsNRT2.3b in improving yield and rice quality.
Background
Rice (Oryza sativa L.) is an important staple food for billions of people (Zeng, et al, 2017). As a big agricultural country, China is one of the earliest countries in the world where rice is planted, and is currently one of the most important food crops in China (Chen Li just, 2019, rime, etc., 2000). For a long time, in order to improve the rice yield, farmers always take fertilization as the most important physicochemical technical measure for rice production. The traditional agriculture of China pays attention to the use of organic fertilizers, has the effects of promoting the yield increase of crops, improving the quality, improving the soil quality and the like, but has the defects of slow fertilizer efficiency, low nutrient content, large application amount, labor cost, poor yield increase effect and the like (Zhoujiang Ming, 2012). Although inorganic fertilizer has the advantages of quick yield increase, high nutrient content, small dosage and the like, the problems of high grain production cost, degraded soil quality, serious agricultural non-point source pollution and the like caused by excessive application of farmers currently exist. The living standard of people is greatly improved, which directly leads to higher and higher requirements of people on rice quality, and the structural contradiction between the quantity and the quality of grains is more obvious, so that a key link of fertilizer is started to solve the phenomenon of strong rice and weak rice (Xunianlong, and the like, 2018).
Among all the essential nutrients of crops, nitrogen is the primary factor limiting plant growth and yield development. Nitrogen (N) is the element which is absorbed by crops in the largest amount from soil, and has important significance on life activities and yield formation of the crops. In recent years, many studies have been made on the absorption system of nitrate nitrogen at a molecular level. Two classes of genes, NRT1 and NRT2, have been found to be associated with the low affinity nitrate nitrogen transport system (LATS) and the high affinity nitrate nitrogen transport system (HATS), respectively. For some high-affinity nitrate transport proteins, chaperonin is required to regulate the activity of the high-affinity nitrate transport proteins, transport proteins such as arabidopsis thaliana (AtNRT2.1), barley (HvNRT2.1) and rice (OsNRT2.1, OsNRT2.2 and OsNRT2.3a) need to have nitrate transport activity in the presence of NAR2 protein, NRT2 family genes generally have 11 to 12 transmembrane times, a rice protoplast subcellular localization experiment shows that OsNRT2.3a and OsNRT2.3b are transmembrane proteins for cytoplasmic membrane localization, and transmembrane structure prediction shows that OsNRT2.3a and OsNRT2.3b both have typical nitrate transport protein transmembrane structures. OsNRT2.3b expresses in both leaves and roots, the expression level is very low, the expression abundance in the leaves is higher relative to the roots, and the expression is not influenced by the external nitrogen concentration and the form.
Disclosure of Invention
The invention aims to provide application of OsNRT2.3b in improving yield and rice quality.
The purpose of the invention can be realized by the following technical scheme:
application of OsNRT2.3b in improving yield and rice quality. The accession number of the OsNRT2.3b gene in the NCBI database is as follows: AK 072215.
The overexpression vector of OsNRT2.3b is applied to improving the rice yield and rice quality.
The OsNRT2.3b overexpression material obtained by the overexpression technology can improve the yield and the rice quality under different fertilizer treatment conditions.
Has the advantages that:
1. through systematic research, the invention discovers for the first time that different fertilizer treatments and OsNRT2.3b overexpression materials have interaction effects of different degrees on agronomic shapes such as rice plant height, effective tillering, biomass, single spike weight, seed setting rate, thousand kernel weight, single spike weight and the like. The method is characterized in that the significant differences of effective tillering, single spike weight and single plant yield of OsNRT2.3b overexpression materials of different fertilizer treatments and rice material interaction are found for the first time; the overground biomass, effective tillering, single spike weight and single plant yield of the OsNRT2.3b super-expression material are obviously higher than that of Nipponbare wild type (WT-N) under different fertilizer treatment conditions (figure 1 and table 1).
2. According to the invention, through systematic research, the interaction effects of different degrees of different fertilizer treatments and the grinding quality (brown rice rate, polished rice rate and whole polished rice rate) of the OsNRT2.3b overexpression material are discovered for the first time. The brown rice rate and the polished rice rate of the total OsNRT2.3b overexpression material are higher than those of Nipponbare wild type (WT-N) under different fertilizer treatment conditions for the first time (Table 2).
3. Through systematic research, the invention discovers the interaction effect of different fertilizer treatments and different degrees of appearance quality (moisture content, length-width ratio, chalky grain rate and chalky degree) of the OsNRT2.3b overexpression material for the first time. It was first found that the aspect ratio of OsNRT2.3b over-expressed material was higher than that of the Japanese clear wild type (WT-N) and the chalkiness particle rate and chalkiness degree were significantly lower than that of the Japanese clear wild type (WT-N) under different fertilizer treatment conditions (Table 3).
4. According to the invention, through systematic research, the interaction effects of different degrees of nutrition and taste quality (protein content, amylose content, alkali elimination value and gel consistency) of different fertilizer treatments and OsNRT2.3b overexpression materials are discovered for the first time. The organic fertilizer is found to be capable of obviously reducing the protein content of the OsNRT2.3b overexpression material for the first time. Under different fertilizer conditions, the protein content and the alkali digestion value of the OsNRT2.3b overexpression material are obviously lower than those of a Nippon Qing wild type (WT-N), the gel consistency is obviously higher than that of the Nippon Qing wild type (WT-N), the amylose content is constant, and the OsNRT2.3b overexpression material is not changed along with the height or type of an external fertilizer. Compared with wild type polished rice, the protein content is high, the hardness of the rice is increased, the viscosity is reduced, and the color is deteriorated; the consistency of the rice is short, the rice is dry and lusterless, hard and loose, the taste is poor, and the rice is cooked and the taste quality is reduced. The cooking taste quality of the OsNRT2.3b super-expression material is obviously higher than that of Nipponbare wild type (WT-N), and the influence of external fertilizer treatment on the cooking taste quality is small. (Table 4).
Drawings
FIG. 1 Effect of different fertilizer treatments on overground biomass of OsNRT2.3b overexpression material in maturation period
The specific implementation scheme is as follows:
example 1 construction of OsNRT2.3b Gene overexpression vector
According to the full-length cDNA sequence (found by NCBI website) of OsNRT2.3b gene, software Primer 5.0 is used for designing and amplifying a full-length ORF Primer, two different enzyme cutting sites of Spe I and Bgl II are respectively designed at the 5' end of the Primer for different expression vectors pCAMBIA1302, and a sense chain is overNRT2.3b-F: 5'-caACTAGTGCTACCACGTGTTGGAGATG-3', antisense strand overNRT2.3b-R: 5'-gaAGATCTGAGCAAACCACCAACAAG-3', the Open Reading Frame (ORF) sequence was amplified using the full-length cloning plasmid of the OsNRT2.3b gene cDNA from National Institute of Agricultural Sciences (NIAS) as a template. And recovering and purifying the PCR product containing the target gene, connecting the PCR product into pMD19-T cloning Vector, performing enzyme digestion verification and sequencing. The plasmid with correct sequencing is cut by the speI and BglII respectively to obtain a gene segment containing a specific cutting site. The target gene fragment is connected into a pCAMBIA1302 vector to obtain a 35S:OsNRT2.3b expression vector, and the OsNRT2.3b expression vector is used for later use after enzyme digestion verification.
Example 2 acquisition and molecular biological identification of OsNRT2.3b overexpression plants
Transgenic T 0 After the seedlings are stained and screened by hygromycin or GUS, an OsNRT2.3b-Ox (35S promoter) transgenic positive 50 strain is obtained, and the obtained transgenic seedlings are transplanted to a field for seed reproduction so as to obtain T 0 And (5) seed generation. In positive lines, 18 OsNRT2.3b-Ox transgenic lines with obvious phenotype difference compared with wild type are obtained, and in order to further determine whether target genes in the lines are enhanced, T is taken 0 And (3) carrying out primary RT-PCR identification on total RNA extracted from transgenic seedling leaves to obtain a plurality of strains with target gene enhanced expression, and selecting the expression enhanced strains as a material for next research according to a detection result, wherein the material is named as O8(35S promoter).
To verify the genetic stability of the transgenic lines, the T of the independent lines harvested separately 0 And (3) carrying out water culture and sprouting on the generation seeds and the wild type seeds, accelerating germination for 2-3 d, and then transferring the seeds into clear water containing hygromycin (25mg/L) for screening for 1 week. After screening, the rice seedlings with relatively consistent growth are transferred into an incubator to be normally cultured for 2 weeks. And (3) taking plant root and leaf samples, quickly putting the plant root and leaf samples into a liquid nitrogen cup, and carrying out semi-quantitative RT-PCR detection after RNA is extracted. The result shows that the enhancement effect of the target gene of the over-expression transgenic line is still strong. We will have performed RT-PCR detected T 1 Planting the seedlings in the soil again to obtain T 1 And (5) seed generation.
Example 3 test design
The test set three fertilizer application levels (M: organic fertilizer treatment, NPK: inorganic fertilizer treatment, NPKM: organic fertilizer), two fertilizer gradients (1: low, 2: high), namely organic low (M1), organic high (M2), inorganic low (NPK1), inorganic high (NPK2), inorganic fertilizer + organic fertilizer low (NPKM1), inorganic fertilizer + organic fertilizer high (NPKM2) for 6 treatments, each treatment is designed with 3 repetitions, 18 cells are counted, and the random cell groups are arranged, each cell area is 3.75M 2 (3m is multiplied by 1.25m), the depth of each district is 1.2 m, and each district is isolated by brick concrete to prevent water and fertilizer exchange between the districts. The nitrogen fertilizer is applied with urea (containing N46%) according to the ratio of base fertilizer to tillering fertilizer to ear fertilizer being 5: 2:3, the organic fertilizer is applied once in the base fertilizer period, and is not applied in the later period. Wherein N in the organic fertilizer is more than or equal to 10 percent,the organic matter content is more than or equal to 20 percent. The calcium superphosphate and the potassium chloride are applied at one time in the base fertilizer period, and are not applied in the later period. The test is carried out according to local routine field management, and the control of the rice long-term flooding diseases, insect pests and weeds is uniformly carried out according to corresponding management cultivation measures of local routine rice machine transplanting cultivation. The rice is planted in a mode of seedling bed seedling raising-transplanting. The fertilizer application rates are given in the table below.
Note 1 field plot experiment treatment setting table
Example 4 measurement of production and seed copy
In the mature period of the rice, 6 rice plants in each cell are randomly investigated, and yield construction factors such as effective spike number, total grain number, single spike weight, maturing rate, thousand kernel weight and the like are examined on the 6 rice ears according to an average effective spike number method. The results are shown in FIG. 1 and Table 1.
Example 5 determination of Rice quality
The harvested paddy is stored for 3 months at room temperature to ensure the stability of the quality of the paddy, and before the determination, the treatment is carried out by air blowing with NP-4350 type air blowing machine and the like. The rice quality refers to the method of GB/T17891-1999 high-quality rice, and the method is used for measuring the brown rice rate, the polished rice rate, the whole polished rice rate, the chalky grain rate, the chalky degree and the like. The crude protein content and amylose content in the polished rice were measured with a near-infrared grain analyzer of the Bowden DA7200, produced in Sweden. The results are shown in tables 2 to 4.
Sequence listing
<110> Nanjing university of agriculture
Application of <120> OsNRT2.3b in improving yield and rice quality
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caactagtgc taccacgtgt tggagatg 28
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gaagatctga gcaaaccacc aacaag 26
Claims (2)
1. OsNRT2.3bThe application in improving the quality of rice; characterized by overexpressionOsNRT2.3bCan improve the grinding quality, appearance quality, nutrition and taste quality of rice.
2.OsNRT2.3bThe super expression vector is applied to improving the quality of rice; it is characterized in thatOsNRT2.3bThe super expression vector is transferred into rice to improve the grinding quality, appearance quality, nutrition and taste quality of the rice.
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| WO2014121436A1 (en) * | 2013-02-05 | 2014-08-14 | Plant Bioscience Limited | Transgenic plants |
| CN108587963B (en) * | 2018-05-02 | 2020-07-28 | 山东农业大学 | L ysinibacillus macrocides screened from manioc waste and application thereof |
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| CN101392257A (en) * | 2008-11-10 | 2009-03-25 | 南京农业大学 | Genetic engineering application of nitrate transport protein genom OsNRT2.3 in Rice |
| CN104995304A (en) * | 2013-02-05 | 2015-10-21 | 植物生物科学有限公司 | Transgenic plants |
| CN106868022A (en) * | 2017-04-13 | 2017-06-20 | 武汉生物工程学院 | Promote nitrogen transporter gene OsNPF2.4b and its application that paddy rice number of productive ear is improved |
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