CN108118062A - Applications of the nitrate anion transporter gene OsNRT1.9a in rice selection and breeding - Google Patents

Abstract

The invention discloses the application of a nitrate transport gene OsNRT1.9a in rice selection and breeding, and belongs to the field of plant genetic engineering. The amino acid sequence of the protein encoded by the OsNRT1.9a gene is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.2. In the present invention, by constructing rice OsNRT1.9a gene overexpression plants and OsNRT1.9a gene interference plants, it is found that by increasing the expression of OsNRT1.9a gene, the number of normal rice tillers can be increased, the number of grains per plant and the yield per plant can be increased. The OsNRT1.9a gene can be used in rice breeding to improve rice yield. The OsNRT1.9a gene has important application value in elucidating the effects of nitrogen on plant growth and development and in improving rice plant type.

Description

Application of Nitrate Transport Gene OsNRT1.9a in Rice Breeding

technical field

The invention belongs to the field of plant genetic engineering, and in particular relates to the application of a nitrate transport gene OsNRT1.9a in rice breeding.

Background technique

China's rice planting area accounts for 20% of the world's total crop planting area, but nitrogen fertilizer application accounts for 37% of the world's total application amount; in 1995, China's nitrogen fertilizer production and use amount reached the first in the world, but nitrogen fertilizer use efficiency is low Low, the amount of nitrogen fertilizer application has increased by 20 times compared with 50 years ago. According to this trend, it is expected to triple again by 2050. Excessive application of nitrogen fertilizer will lead to ecological pollution such as eutrophication of water [Xu Guohua, Fan Xiaorong. Functional study of rice nitrate transporter genes OsNRT1.1a and OsNRT1.1b. Nanjing Agricultural University, 2011: 4-6]. More nitrogen nutrients are wasted through denitrification, soil erosion, natural volatilization, and microbial utilization.

A 1% increase in nitrogen uptake efficiency equates to an annual savings of more than one billion dollars. From the analysis of China's national conditions, the potential for expanding the planting area to increase the total output is very limited. The only way out is to produce more rice on limited land, that is, to increase the output per unit area. In the past traditional farming, nitrogen use efficiency was improved by selecting crops with higher nitrogen use efficiency; but compared with molecular breeding, this process was slow and inefficient [Zhang Hongcheng, Dai Qigen. Nitrogen Utilization in Rice Genotype differences and physiological mechanism research. Yangzhou University, 2008: 10-13]. To improve nitrogen utilization, a breakthrough must be found in the molecular absorption mechanism of nitrogen. The nitrate transport gene family is divided into low-affinity nitrate transport genes and high-affinity nitrate transport genes [Zhou Shiyi. Effects of sugars and amino acids on rice-induced high-affinity nitrate transport system. Huazhong University of Science and Technology, 2009: 15- 16]. Through nitrogen assimilation, nitrate nitrogen and ammonium nitrogen are absorbed and converted into amino acids, which is called the first type of nitrogen absorption. Through the transport of nitrogen, the nutrients of seeds are increased, and the plumpness is increased, which is called the second type of nitrogen absorption, that is, the reuse of nitrogen [Kant S, Bi Y, Steven J, etal. Understanding plant response to nitrogen limitation for the Improvement of crop nitrogen use efficiency. Journal of Experimental, 2011, 62(4): 1499-1509]. Increasing nitrogen uptake and accumulation or nitrogen transport can increase yield. Therefore, in the construction of modern agriculture, improving the nitrogen utilization efficiency of rice through molecular breeding can reduce nitrogen pollution and increase yield.

NRT1/PTR family (NRT1/PTR family, NPF) refers to the protein that can mediate the transmembrane transport of substances such as small molecular peptides and nitrate radicals with 2-3 amino acid residues [Rentsch D, Schmidt S, Tegeder M. Transporters for uptake and allocation of organic nitrogen compounds in plants. FEBS Let, 2007, 581: 2281-2289]. NRT1/PTR family members are involved in the accumulation of proteins during seed formation and the transport of small molecule polypeptides after protein degradation during germination [Martre P, Porter J R, Jamieson P D, et al. Modelinggrain nitrogen accumulation and protein composition to understand the sink/source regulations of nitrogen remobilization for wheat. Plant Physiol, 2003, 133:1959-1967]. At present, there are few reports on the research on the members of the NPF family. The OsNRT1.9 gene involved in the present invention is a nitrate transport gene of the rice NPF gene family. The present invention finds that post-transcriptional processing of the OsNRT1.9 gene can form two kinds of splicing, wherein the first splicing form OsNRT1.9a has an extremely important effect on rice tillering, and can be applied to plant type improvement to increase rice yield.

Contents of the invention

The purpose of the present invention is to solve the problems existing in the prior art, and to provide the application of the rice NPF gene family member nitrate transport gene OsNRT1.9a in rice selection and breeding.

The object of the present invention is achieved through the following technical solutions:

The invention takes the rice NPF gene family member nitrate transport gene OsNRT1.9a as object, and clones the cDNA sequence of OsNRT1.9a from rice Zhonghua 11. By constructing the OsNRT1.9a gene overexpression vector, using the genetic transformation method mediated by Agrobacterium EHA105, the overexpression vector was introduced into the normal japonica rice variety Zhonghua 11, and the OsNRT1.9a gene overexpression plants were obtained. Compared with the control wild type Zhonghua 11, the number of grains, grain filling and yield were significantly increased. The OsNRT1.9a gene interference expression vector was constructed by RNAi technology, and the interference expression vector was introduced into Zhonghua 11 to obtain the interference plants with decreased OsNRT1.9a gene expression. Compared with Zhonghua 11, it was significantly lower. These results indicated that by increasing the expression of the OsNRT1.9a gene, the number of tillers in normal rice can be increased, thereby increasing the number of panicles, the number of grains at filling and rice yield.

Based on the function of the OsNRT1.9a gene discovered in the present invention, the OsNRT1.9a gene can be used in rice breeding. The rice breeding is to increase the number of rice tillers, thereby increasing the number of panicles, the number of grains for filling and the yield of rice. Specifically, by increasing the expression of the OsNRT1.9a gene, the number of rice tillers, the number of spikes per plant, and the number of grains filling can be increased, so as to achieve the purpose of increasing rice yield.

The OsNRT1.9a gene can also be used to increase the yield of other plants, for example, the (over)expression of the OsNRT1.9a gene in the plant through transgenics can increase the number of branches of the plant, thereby increasing the yield of the plant. The plants refer to monocotyledonous plants or dicotyledonous plants; such as: wheat, tomato, lawn grass or alfalfa.

The amino acid sequence of the OsNRT1.9a protein encoded by the OsNRT1.9a gene is shown in SEQ ID NO.1; the cDNA sequence of the OsNRT1.9a gene is preferably shown in SEQ ID NO.2.

It should be understood that, on the premise of not affecting the activity of OsNRT1.9a (that is, not in the active center of the protein), those skilled in the art can make various substitutions, additions and/or deletions to the amino acid sequence shown in SEQ ID NO.1 One or several amino acids obtain amino acid sequences with equivalent functions. Therefore, the OsNRT1.9a protein also includes proteins with equivalent activity obtained by substituting, substituting and/or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1. In addition, it should be understood that, considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for the expression of specific species as needed.

Advantages and effects of the present invention:

(1) After the overexpression of the OsNRT1.9a gene cloned in the present invention, the rice tillering ability is enhanced, indicating that the OsNRT1.9a gene is more obvious for improving the rice yield. Therefore, improving the expression of the OsNRT1.9a gene through genetic engineering technology can increase the plant yield. This not only helps to breed high-yielding rice under normal nitrogen fertilization conditions, but also enables plant variety improvement through molecular breeding.

(2) The successful cloning of the OsNRT1.9a gene further confirmed the important role of the NPF family in the process of nitrogen uptake, which is of great significance for elucidating the biological functions of the NPF family, and for further understanding of plant nitrogen metabolism pathways and improving nitrogen uptake Efficiency has a huge boost.

(3) Although some genes that increase plant yield have been cloned, the molecular mechanism of plant yield increase is still unclear. However, the OsNRT1.9a gene cloned in the present invention can increase the yield of rice, and has a great role in promoting the determination of key factors for plant yield increase.

Description of drawings

Fig. 1 is the whole plant phenotype diagram of 3 lines of control Zhonghua 11 and OsNRT1.9a gene overexpressed plants.

Figure 2 is a statistical histogram of the number of tillers in three strains of the control Zhonghua 11 and OsNRT1.9a gene overexpression plants. The data was analyzed by SPSS software (ANOVA), using Duncan's at three levels of 0.05, 0.01 and 0.001 Significant difference analysis, compared with the control, respectively represented by *, ** and ***.

Fig. 3 is a diagram of the whole plant phenotype of three lines of the control Zhonghua 11 and OsNRT1.9a gene interference plants.

Figure 4 is a statistical histogram of the number of tillers in the three strains of the control Zhonghua 11 and OsNRT1.9a gene interference plants. The data was analyzed by SPSS software (ANOVA), and Duncan's was used to make differences at three levels: 0.05, 0.01 and 0.001 Significant analysis, compared with the control, respectively represented by *, ** and ***.

Figure 5 is a graph showing the detection results of OsNRT1.9a gene expression in the control Zhonghua 11, 3 lines of OsNRT1.9a gene overexpression plants and 3 lines of OsNRT1.9a gene interference plants, and the data were analyzed by SPSS software (ANOVA ), using Duncan's at three levels of 0.05, 0.01 and 0.001 to carry out significant difference analysis, compared with the control, represented by *, ** and *** respectively.

Fig. 6 is a diagram of the grain filling phenotype of each plant of control Zhonghua 11, 3 lines of OsNRT1.9a gene overexpression plants and 3 lines of OsNRT1.9a gene interference plants.

Fig. 7 is a statistical chart of the number of grains per plant of the control Zhonghua 11, 3 lines of OsNRT1.9a gene overexpression plants and 3 lines of OsNRT1.9a gene interference plants. The data were analyzed by SPSS software (ANOVA), and Duncan’s was used to conduct significant difference analysis at three levels of 0.05, 0.01 and 0.001, and compared with the control, they were represented by *, ** and ***, respectively.

Fig. 8 is the yield statistical chart of each plant of the control Zhonghua 11, 3 lines of OsNRT1.9a gene overexpression plants and 3 lines of OsNRT1.9a gene interference plants. The data are analyzed by SPSS software (ANOVA), using Duncan's Significant difference analysis was carried out at three levels of 0.05, 0.01 and 0.001, which were represented by *, ** and *** respectively compared with the control.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. If not otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental methods used are conventional methods, and can be described in accordance with recombinant techniques (referring to molecular cloning, laboratory manual, The second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) was completed; the materials and reagents used can be obtained from commercial channels.

Example 1 Construction of OsNRT1.9a Gene Overexpression Plants

Extract the RNA of rice Zhonghua 11 and reverse transcribe it into cDNA, using the primer pair:

F1: 5'- AGATCT ATGGCCGCCATAGAAGAGGAG-3' (Bgl II),

R1: 5'- CTTAAG TCATGAAGCTGTGTTTCTCTCT-3' (Afl II);

After the cDNA of the OsNRT1.9a gene was amplified by PCR, it was digested with Bgl II and Afl II and then ligated into the pCAMBIA-1301 vector (the pCAMBIA-1301 vector was purchased from Cambia Company) to construct the overexpression vector OsNRT1 of the OsNRT1.9a gene. 9a-p1301. Using the genetic transformation method mediated by Agrobacterium EHA105, the overexpression vector was introduced into the normal rice variety Zhonghua 11.

Transplant all the obtained transgenic seedlings into baskets with soil, water and fertilize them regularly, and plant them in the field when the seedlings grow about 10cm in height. After the seedlings grow up, extract genomic DNA and detect the transgenic plants by PCR , the detection primer pair is:

F2: 5'-GATGTTGGCGACCTCGTATT-3',

R2: 5'-TCGTTATGTTTATCGGCACTTT-3'.

If a 517bp fragment is amplified, it indicates that the transgenic plant is a positive plant. Positive plants were harvested and planted until homozygous transgenic plants were identified in the T2 generation, and OsNRT1.9a gene overexpressed plants were obtained. The number of tillers in the OsNRT1.9a gene overexpressed plants was much more than that of the control Zhonghua 11 plants, and the difference was significant, as shown in Figures 1 and 2. Detection of the expression level of the OsNRT1.9a gene in the overexpressed plants showed that the expression of the OsNRT1.9a gene was increased compared with the control, as shown in FIG. 5 . Seed statistics were collected from a single plant, and the results showed that the grain filling per plant of the overexpression plant increased, and the yield per plant increased, as shown in Figures 6, 7 and 8.

Example 2 Obtaining of OsNRT1.9a gene interference plants

Extract the RNA of rice Zhonghua 11 and reverse transcribe it into cDNA, using the primer pair:

F3: 5'- GGTACC AGCCAGCCCTGAAGCACAGCAC-3' (Kpn I),

R3: 5'- GGATCC TGCACCACTCCCAAGGGCAGCA-3' (BamH I);

F4: 5'- ACTAGT AGCCAGCCCTGAAGCACAGCAC-3' (Spe I),

R4: 5'- GAGCTC TGCACCACTCCCAAGGGCAGCA-3'(Sac I);

After the cDNA fragments of the OsNRT1.9a gene were amplified by PCR, they were digested with corresponding restriction enzymes and then ligated into the pTCK303 vector to construct the interference expression vector OsNRT1.9a-pTCK303 of the OsNRT1.9a gene. Using the genetic transformation method mediated by Agrobacterium EHA105, the interference expression vector was introduced into the normal japonica rice variety Zhonghua 11.

Transplant all the obtained transgenic seedlings into baskets with soil, water and fertilize them regularly, and plant them in the field when the seedlings grow about 10cm in height. After the seedlings grow up, extract genomic DNA and detect the transgenic plants by PCR , the detection primer pair is:

F2: 5'-GATGTTGGCGACCTCGTATT-3',

R2: 5'-TCGTTATGTTTATCGGCACTTT-3'.

If a 517bp fragment is amplified, it indicates that the transgenic plant is a positive plant. Positive plants were harvested and planted until a homozygous transgenic plant was identified in the T2 generation, and the OsNRT1.9a gene interference plant was obtained. The number of tillers of OsNRT1.9a gene interference plants was far less than that of the control Zhonghua 11 plants, and the difference was significant, as shown in Figures 3 and 4. Detection of the expression level of the OsNRT1.9a gene in the disturbed plants showed that the expression of the OsNRT1.9a gene was reduced compared with the control, as shown in FIG. 5 . Seed statistics were collected from a single plant, and the results showed that the grain filling per plant of the interfering plants decreased, and the yield per plant decreased, as shown in Figures 6, 7 and 8.

The above results indicated that by increasing the expression of OsNRT1.9a gene, the tiller number of rice can be increased, thereby increasing the panicle number and rice yield.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

sequence listing

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Claims (10)

1. The application of OsNRT1.9a gene in rice breeding, characterized in that: the rice breeding is to increase the tiller number of rice. 2. The application of OsNRT1.9a gene in increasing the panicle number of rice. 3. Application of OsNRT1.9a gene in increasing rice grain number. 4. Application of OsNRT1.9a gene in improving rice yield. 5. Application of OsNRT1.9a gene in improving plant yield. 6. The application according to any one of claims 1-5, characterized in that: the application is realized by increasing the expression of OsNRT1.9a gene. 7. The application according to claim 5, characterized in that: said plants refer to monocotyledonous plants or dicotyledonous plants. 8. The application according to claim 5 or 7, characterized in that: said plants include wheat, tomato, lawn grass or alfalfa. 9. The application according to any one of claims 1-5, characterized in that: the amino acid sequence of the OsNRT1.9a protein encoded by the OsNRT1.9a gene is shown in SEQ ID NO.1; or the OsNRT1.9a protein A protein with equivalent activity obtained by substituting, substituting and/or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1. 10. The application according to claim 9, characterized in that: the cDNA sequence of the OsNRT1.9a gene is shown in SEQ ID NO.2.