CN110551719B

CN110551719B - Long-chain non-coding RNA gene ALEX1 and application thereof in improving bacterial leaf blight resistance of rice

Info

Publication number: CN110551719B
Application number: CN201910693239.4A
Authority: CN
Inventors: 于洋; 周燕飞; 张玉婵; 陈月琴
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2023-06-02
Anticipated expiration: 2039-07-30
Also published as: CN110551719A

Abstract

The invention discloses a long-chain non-coding RNA gene ALEX1 and application thereof in improving bacterial leaf blight resistance of rice. The invention clones long-chain non-coding RNA ALEX1 gene from rice, and the nucleotide sequence is shown in SEQ ID NO:1, the transcription product is induced by bacterial leaf blight bacteria to up-regulate expression. Biological function research on ALEX1 proves that activating the expression can improve the content of jasmonic acid in rice and obviously enhance the resistance of the rice to bacterial blight, and the long-chain non-coding RNA ALEX1 can positively regulate the resistance of the rice to bacterial blight. The invention provides a new strategy and genetic resource for cultivating new rice disease-resistant varieties, in particular new rice bacterial leaf blight-resistant varieties, and has very important theoretical significance and application value.

Description

Long-chain non-coding RNA gene ALEX1 and application thereof in improving bacterial leaf blight resistance of rice

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a long-chain non-coding RNA gene ALEX1 and application thereof in improving bacterial leaf blight resistance of rice.

Background

Plant diseases seriously affect the yield and quality of crops, and the cultivation of disease-resistant plants is one of important subjects of crop molecular breeding research. Bacterial leaf blight of rice caused by pathogenic strain Xoo (Xanthomonas oryzae pv. Oryzae) of gram-negative bacteria Xanthomonas is one of the most common diseases in rice production, pathogenic bacteria invade through water holes or wounds of rice leaves, and a large amount of polysaccharide substances are propagated and secreted in vascular bundles to block the vascular bundles, so that the leaves are wilted and even dead, photosynthesis of rice is seriously reduced, and yield reduction and even harvest failure of rice are caused.

At present, a plurality of rice bacterial leaf blight resistance related genes have been identified internationally, especially by scientists in China, but cloning and functional research of the genes are relatively few. On the other hand, bacterial leaf blight pathogenic strains are continuously mutated under the double pressures of natural selection and manual selection, so that further deep excavation of rice genetic resources with bacterial leaf blight resistance has very important theoretical significance and application value for crop genetic improvement and solving of grain safety problems.

Long non-coding RNAs (lncRNA) are a class of non-coding RNAs with a length greater than 200nt, which are widely involved in various aspects of organism development and metabolism, another research hotspot behind small-molecule RNAs in the field of non-coding RNA research, and the molecular mechanism of lncRNA to regulate plant growth and development and crop agronomic traits has become a significant scientific frontier problem in the field of life science. Several lncRNA functions have been reported in mammals, demonstrating that lncRNA can be involved in the phylogenetic development, immune regulation, and the occurrence and progression of diseases in mammals. However, the studies on the lncRNA in plants are relatively few, and the application of the lncRNA on the disease resistance and the defense response of the lncRNA and the bacterial leaf blight of rice is blank so far.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings in the prior art and provide a long-chain non-coding RNA gene ALEX1 related to the bacterial leaf blight resistance of rice, which can obviously improve the resistance of the rice to bacterial leaf blight. The invention has important application value for molecular genetic breeding of important grain crop disease-resistant varieties such as rice and the like.

The second object of the present invention is to provide the application of the long-chain non-coding RNA gene ALEX1 in improving bacterial leaf blight resistance of rice.

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

a long-chain non-coding RNA ALEX1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The research of the invention discovers that the long-chain non-coding RNA (lncRNA) is specifically expressed only in the leaf after bacterial leaf blight infection and hardly expressed under normal physiological conditions, and the long-chain non-coding RNA is named ALEX1. The 5'-RACE and 3' -RACE have proved that the ALEX1 gene is located on the antisense DNA strand of rice chromosome 8, the gene covers the range from 22381757bp to 22382050bp, lncRNA with the length of 294nt can be transcribed, and the nucleotide sequence is shown as SEQ ID NO. 1. The promoter sequence of the long-chain non-coding RNA ALEX1 gene is shown as SEQ ID NO. 2.

The invention also provides a pair of primer pairs which can be used for amplifying the full-length DNA sequence of the ALEX1 gene, wherein the primer pairs comprise an upstream primer and a downstream primer, and the nucleotide sequences of the primer pairs are shown as SEQ ID NO. 3-4 in sequence:

F：5’-CCGGCGACAAGATTTGCAGAG-3’(SEQ ID NO:3)；

R：5’-CGCCGTGACATCACCTGGCAATGG-3’(SEQ ID NO:4)。

the present invention demonstrates that in situ expression activated plants (ALEX 1ox-1 and ALEX1 ox-2) based on the ALEX1 promoter enhance rice resistance to bacterial blight. It was found that significant changes in expression of JA signaling pathway-related genes (e.g., JAZ8, MYC2, PR1a, PR1b, PR10a, RSOsPR10, etc.) in ALEX1ox-1 occurred; compared with WT, the root of ALEX1ox-1 is shorter, and the gradient concentration treatment of MeJA also shows that ALEX1ox-1 is more sensitive to MeJA, and the endogenous hormone content measurement result shows that the content of JA and JA-Ile in the expression activated plant of ALEX1 is obviously increased. These results together indicate that the JA signal is activated in rice ALEX1 high expression plants, suggesting that ALEX1 may be involved in JA-mediated defense responses of rice against bacterial leaf blight.

Thus, the present invention also provides an in situ activating expression vector comprising necessary elements for targeting the promoter region of the ALEX1 gene and recruiting transcriptional activators to promote the expression level of the gene itself, enhancing the expression level of the ALEX1 gene at the in situ level; the promoter sequence of the ALEX1 gene is shown as SEQ ID NO. 2.

Meanwhile, the invention constructs an ALEX1 overexpression vector UBI under the drive of a UBI promoter based on the whole sequence length of the ALEX1 shown in SEQ ID NO. 1, and transforms the expression vector into rice callus through agrobacterium tumefaciens to obtain transgenic rice plant OXALEX1 which overexpresses the ALEX1. Experimental results prove that in three OXALEX1 transgenic rice lines, the expression of related genes of a jasmonic acid synthesis pathway or a signal pathway is obviously up-regulated, and the resistance of the jasmonic acid synthesis pathway or the signal pathway to bacterial leaf blight is also obviously improved.

Therefore, the invention also provides a recombinant overexpression vector, which contains the ALEX1 gene.

Preferably, the recombinant overexpression vector contains a UBI promoter sequence.

The invention also protects a host bacterium containing the in-situ activated expression vector or the recombinant super-expression vector.

Thus, the following applications based on the above research results are all within the scope of the present invention:

the application of the long-chain non-coding RNA ALEX1 gene, the in-situ activating expression vector, the recombinant super-expression vector or the host bacteria in improving the content of jasmonic acid and/or jasmonic acid-isoleucine complex in rice.

The long-chain non-coding RNA ALEX1 gene, the in-situ activating expression vector, the recombinant super-expression vector or the host bacteria are applied to improving the bacterial leaf blight resistance of rice.

Specifically, the application is to activate expression of long-chain non-coding RNA ALEX1 genes in rice in situ or to transform the long-chain non-coding RNA ALEX1 genes into rice by constructing an overexpression vector of the long-chain non-coding RNA ALEX1 genes; namely a method for improving the bacterial leaf blight resistance of rice: activating expression of long-chain non-coding RNA ALEX1 gene in rice in situ or transforming the rice into rice by constructing super-expression vector of long-chain non-coding RNA ALEX1 gene.

The long-chain non-coding RNA ALEX1 gene, the in-situ activating expression vector, the recombinant super-expression vector or the host bacteria are applied to cultivation of rice varieties resistant to bacterial leaf blight.

The promoter sequence shown in SEQ ID 2 and based on the rice long-chain non-coding RNA gene ALEX1 is applied to improving the content of jasmonic acid-isoleucine complex in rice or enhancing the resistance of rice to bacterial leaf blight.

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

the invention provides a long-chain non-coding RNA gene ALEX1 and application thereof in improving bacterial leaf blight resistance of rice, the invention clones the long-chain non-coding RNA ALEX1 gene from the rice, and the nucleotide sequence is shown as SEQ ID NO:1, the transcription product is induced by bacterial leaf blight bacteria to up-regulate expression. Biological function research on ALEX1 proves that activating the expression can improve the content of jasmonic acid in rice and obviously enhance the resistance of the rice to bacterial blight, and the long-chain non-coding RNA ALEX1 can positively regulate the resistance of the rice to bacterial blight. The invention discloses reports about the influence of long-chain non-coding RNA on the jasmonic acid signal of rice or the capability of regulating and controlling bacterial leaf blight disease resistance for the first time, provides a new strategy and genetic resource for cultivating new rice varieties with disease resistance, in particular to new rice varieties with bacterial leaf blight resistance, and has very important theoretical significance and application value.

Drawings

FIG. 1 is a cluster analysis and expression level verification of bacterial leaf blight response lncRNA. (a) Thermal mapping of differentially expressed lncRNA in P.sub.XO 99A inoculated rice leaves we used p <0.05, absolute value of expression change >2 as the threshold for significant differential expression analysis. (b) The lncRNA expression profile in RNA-seq was verified by quantitative real-time PCR. The rice gene ACTIN2 was used as an internal reference. The expression level of xoo_0h was set to 1 and the data represent the mean ± standard deviation of three replicates.

FIG. 2 is a GO enrichment and KEGG pathway analysis of Cis targets differentially expressing lncRNA. (a) The GO entries of 512 annotated genes in the GO database were analyzed and enriched in the "cellular component" and "biological process" entities. The enrichment factor was calculated by the number of genes mapped to the GO term, with log10 (FDR) values representing the significance of the GO term. (b) KEGG pathway enrichment analysis of 704 genes, showing the first 20 enrichment pathways with-log 10 on the x-axis (p-value), the significance of KEGG pathway enrichment.

FIG. 3 shows the insertion sites and expression analysis of ALEX 1-activated expression plants. (a) Enhancer traps insert models of gene distribution within a 10kb region upstream or downstream of the site. (b) expression level of lncRNA near the insertion site. RT-PCR was used to detect the expression of XLC_418473, XLC_437332 and ALEX1. Rice ACTIN2 was used as an internal reference.

FIG. 4 shows the expression of ALEX1 and the full-length detection of the sequence. (a) ALEX1 was induced to express at 6h and 12h after bacterial infection, RT-PCR was used to detect ALEX1 expression, and rice ACTIN2 was used as an internal control. (b) 5' -RACE peak assay. (c) 3' -RACE peak assay. (d) full length 294nt sequence of ALEX1.

FIG. 5 shows the detection of resistance of ALEX1 in situ expression activated plants to bacterial leaf blight of rice. (a) Comparison of resistance to Xoo of wild type and ALEX1 activated expression plants rice flag leaves were inoculated with PXO99A and photographed after 14 days; (b) Leaf spot length (n=30) of wild type and ALEX1 activated expression plants after 4 weeks of PXO99A infection, asterisks indicate statistical differences (< 0.001) compared to wild type after t-test; (c) Real-time fluorescent quantitative PCR analysis of the relative DNA content between HrpC of Xoo and rice EF-1. Alpha. In wild type and ALEX1ox-1 activated expression plants, the data are the mean.+ -. Standard deviation of triplicates.

FIG. 6 shows detection of the activation of jasmonic acid signal regulation in plants by ALEX1 in situ expression. (a) Relative expression levels of the output genes in JA signaling of ALEX1ox-1 and WT plants, ACTIN2 was used as an internal reference, data represent mean ± standard deviation of triplicate, asterisks indicate statistically significant differences compared to wild type by t-test (< 0.01;: < 0.001: < P); (b) MeJA treatments with different concentration gradients are carried out on rice seedlings after germination for 3 days, and changes of ALEX1ox-1 and WT plant root system development after the treatment for 3 days are shown in the diagram, wherein the scale is=2 cm; (c) The levels of endogenous jasmonic acid in ALEX1ox-1 and WT plants were determined using LC-MS/MS for the concentrations of JA, JA-Ile and OPDA, data expressed as mean ± standard deviation of triplicate, asterisks indicate statistical differences compared to wild type by t-test (< 0.05;: < 0.001).

FIG. 7 is a phenotypic analysis of ALEX1 overexpressing plants. (a) RT-PCR (reverse transcription-polymerase chain reaction) detection of the expression effect of ALEX1 in 10 ALEX1 over-expression plants; (b) a plaque phenotype after 10 days of bacterial infection; (c) Spot length statistics, n=15, asterisks indicate statistically significant differences compared to wild type by t-test (< P < 0.001).

FIG. 8 shows the expression level of the JA-associated gene in ALEX1 overexpressing plants. ACTIN2 was used as an internal control, data represent mean ± standard deviation of triplicate, asterisks indicate statistically significant differences compared to wild type by t-test (< 0.01; < 0.001).

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified. The primer sequences used were all synthesized by the biological technology company of the family borreliaceae, beijing.

Example 1 screening of differential expression of Rice lncRNA after bacterial leaf blight infection

To study the role of lncRNA in the disease-resistant response of rice, the inventors performed strand-specific lncRNA sequencing on rice leaf samples infected with bacterial leaf blight pathogen PXO99A for 0, 2h, 6h, 12h and 24h, resulting in 5.7X10 ⁸ reads, 4.9X10 ⁸ reads matched the reference genome of rice and accounted for 85.26% of the total data obtained. 70 ten thousand transcripts are obtained after splicing and assembling, and 48727 rice lncRNA transcripts with higher credibility are finally obtained through 5 basic screening processes and coding potential screening. Quantitative analysis was performed on lncRNA obtained by screening using cuffdiff software to obtain FPKM information of lncRNA of each sample, and the expression level of 567 lncRNA was significantly changed due to infection with bacterial leaf blight bacteria (fig. 1). To confirm the reliability of the deep sequencing data of the lncRNA expression profile, we selected 8 lncRNA with different expression patterns and verified their expression by qRT-PCR, the results were consistent with the results obtained from lncRNA sequencing, indicating that the sequencing dataset was reliable (fig. 1). Regarding cis mRNA (from 704 genes in total) which is expressed in correlation with the 567 differentially expressed lncRNAs and has adjacent gene loci, performing GO enrichment analysis and KEGG pathway analysis, we find that the defense reaction of rice and the metabolic pathway of alpha-Linolenic acid (synthetic raw materials of jasmonic acid compounds) have obvious enrichment effect (figure 2), and suggest that the lncRNAs may have important functions in bacterial leaf blight infection of rice and JA-mediated disease resistance regulation.

Example 2 acquisition and functional identification of ALEX1 in situ expression activated plants

The 567 bacterial leaf blight-responsive lncRNAs identified above are candidate genetic resources for regulating resistance of rice to bacterial leaf blight. To investigate whether abnormal expression of these lncRNAs would affect the disease course of bacterial leaf blight of rice, we searched the global major rice mutant database and identified that an activation expression plant of lncRNA exhibited significant bacterial leaf blight resistance (FIG. 5). Since the lncRNA is only highly expressed in leaf blades after bacterial infection and hardly expressed under normal physiological conditions (FIG. 4), we named ALEX1 (An LncRNA Expressed in Xoo-infected leaves), and two active expression lines of ALEX1 were named ALEX1ox-1 and ALEX1ox-2.

We have confirmed that ALEX1 is located on the antisense DNA strand of chromosome 8 of rice by 5'-RACE and 3' -RACE, and that the ALEX1 gene covers a range from 22381757bp to 22382050bp, which can transcribe lncRNA of 294nt in length (FIG. 4). The insertion sites of ALEX1ox-1 and ALEXox-2 constructed by using the enhancer capture system are located 60bp upstream of ALEX1 and belong to the promoter region of the gene, and experimental results show that the enhancer trap insertion of the sites has the effect of enhancing the expression of ALEX1 (figure 3). To further clarify the correspondence between resistance of the transgenic plant to bacterial blight and ALEX1 activation expression, we searched for genes near the insertion site of the ALEX1 enhancer trap in detail, and found that there were only 3 lncRNA sites within the 10kb region of the insertion site, without any protein coding genes (fig. 3). We then studied the expression levels of these three lncRNAs by RT-PCR. XLOC_418473 and XLOC_437332 were not detected for expression in either ALEX1ox-1 or wild type, whereas ALEX1 was not expressed in WT, but significantly upregulated in ALEX1ox-1 (FIG. 3). The above results indicate that insertion of the enhancer capture system into this site only enhances expression of ALEX1, and that resistance of ALEX1ox-1 to bacterial leaf blight is due to cumulative expression of ALEX1, but not to changes in expression of any other regulatory factors in the vicinity of the insertion site.

Next, we examined the expression of jasmonic acid signal pathway related genes in wild type and ALEX1 activated expression strains, found that the mRNA expression levels of important regulatory factors such as JAZ8, MYC2, PR1a, PR1b, PR10a and RSOsPR10 were significantly changed, and the elongation of roots of ALEX1ox-1 plants was also significantly inhibited, and the endogenous hormone content measurement result also showed that the content of jasmonic acid and its active form jasmonic acid-isoleucine in the expression activated plants of ALEX1 was significantly increased (fig. 6). The primer sequences used are shown in Table 1:

TABLE 1 expression primers for jasmonic acid signal pathway related genes

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Example 3 ALEX1 in situ expression activated plant exogenous methyl jasmonate treatment and determination of endogenous jasmonate content

Wild medium flower No. 11 and ALEX1 expression in situ activated rice seed germination in water at 32 ℃ for 3 days. Methyl jasmonate (Sigma Aldrich # 392707) was diluted to the indicated concentration in deionized water and used to treat the 3-d-old seedlings described above. The root state was photographed after 6 days of cultivation in a climatic chamber at 27℃and 70% humidity. Methyl jasmonate treatment experiments showed that JA signal was enhanced in rice ALEX1 in situ expression activated plants (fig. 6).

JA. JA-Ile and OPDA were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Briefly, the highest leaf of 3 week old rice was homogenized in liquid nitrogen. A 200mg sample of powder of fresh tissue was added to a sample containing isopropanol: water 1,000. Mu.L of HCl in 500:1 extraction buffer, with an internal standard of 10ng H2JA (Cerelliant). The mixture was stirred at 4℃for 30 minutes, then 1ml of CH was added ₂ Cl ₂ Gently stir for 30 minutes. After centrifugation at 13,000Xg for 10 minutes, the lower layer was collected at 4℃with about 900. Mu.L of solvent; to the sample, 100. Mu.L of 60% methanol was added, and 10. Mu.L of each was injected into the C18 column for further measurement by LC-MS/MS (AB Sciex Q-TOF 5600+ system). The endogenous hormone content measurement results showed that expression of ALEX1 activated the levels of JA and JA-Ile in the plants to be significantly increased (fig. 6). This isThe results comprehensively show that the JA signal is activated in rice ALEX1 high-expression plants, which suggests that the ALEX1 possibly participates in the JA-mediated defense reaction of rice against bacterial leaf blight.

EXAMPLE 4 construction, transformation and disease resistance phenotype analysis of ALEX1 overexpressing plants

The total RNA of the middle-colored rice No. 11 after being infected by wild type or bacterial leaf blight bacteria is amplified to obtain the full-length DNA sequence 294bp of ALEX1,

the primer F sequences used were:

5’-GGACTAGTCCGGCGACAAGATTTGCAGAG-3’，

the primer R sequences used were:

5’-ATAAGAATGCGGCCGCCGTGACATCACCTGGCAATGG-3’，

SpeI and NotI were used for cleavage sites.

Constructing an ALEX1 overexpression vector guided by a UBI promoter by adopting pRHV plasmid presented by a national beam researcher of China academy of agricultural science; transgenic treatment is carried out by using an agrobacterium-mediated callus genetic transformation method, and corresponding vectors are transferred into rice; transformants were screened with hygromycin (Hyg) to obtain transgenic rice plants over-expressed by ALEX1.

In order to determine the disease resistance of different rice strains to bacterial leaf blight, a leaf-cutting inoculation method is adopted for infection. The disease resistance of the rice is determined by measuring the degree of the disease spots. In the process, the bacterial wilt and different rice strains are prepared at the same time, and the disease resistance of the rice in the booting stage is measured.

Preparation of rice: and (3) planting the rice outdoors, wherein the distance between each row is 25cm, the plant spacing is 20cm, and inoculating the white leaf blight bacteria when the rice grows to the booting stage. Activating the bacterial leaf-blight on a PDA solid culture medium, transferring the activated bacterial leaf-blight into a liquid culture medium, culturing until the OD600 = 0.8-1.0, centrifuging at 3000rpm for 1min, collecting bacterial cells, washing twice with sterile water, and regulating the OD600 = 0.2 for later use.

Infection experiment: immersing the head of the clean sterilized scissors into bacterial liquid, sticking the bacterial liquid, and then transversely cutting the leaves at the position of 2-3 cm below the tips of the fully-unfolded leaves. The leaves were then allowed to develop for two weeks.

Measuring the lesions: and measuring the length from the leaf tip to the bottom end of the disease spot, and measuring the length of the leaf. Photographing: the infected leaves are taken out, stuck to white paper by double-sided tape, and then photographed.

Detection index of bacterial growth rate: infected leaves approximately 20cm long were collected 0,3 and 6 days after infection. DNA samples were extracted and the bacterial hrpC and rice EF-1. Alpha. Gene copy numbers were quantified using qRT-PCR using the primer sequences shown in Table 2 below:

TABLE 2 expression primers for genes of bacterial hrpC and rice EF-1 alpha

Primer name	Sequence (5 '. Fwdarw.3')
		EF1a-F	CTGGACTGCCACACCTCACACAT
EF1a-R	CCAACAGCCACCGTTTGCCTC
		HrpC-F	GGGGTGTCGTTGCGGGTATT
HrpC-R	ATCGGTCTCGTCGGCATCGTA

The above experimental detection of ALEX1 over-expressed plants further proves that the long-chain non-coding RNA ALEX1 plays an important role in regulating the resistance of rice to bacterial leaf blight (FIG. 7 and FIG. 8).

Sequence listing

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Claims

1. The application of a recombinant overexpression vector containing a long-chain non-coding RNA ALEX1 gene in improving bacterial leaf blight resistance of rice is disclosed, wherein the nucleotide sequence of the ALEX1 gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein said use is the transformation of a recombinant overexpression vector of the long-chain non-coding RNA ALEX1 gene into rice.

3. The application of the recombinant super-expression vector containing the long-chain non-coding RNA ALEX1 gene in cultivating rice varieties resistant to bacterial leaf blight is disclosed, and the nucleotide sequence of the ALEX1 gene is shown as SEQ ID NO. 1.