Flax gene LuWRI1a and its application in improving salt and drought tolerance

Abstract

The invention discloses a flax gene LuWRI1a and an application thereof in improving the salt and drought resistance of plants, belonging to the technical field of genetic engineering. The nucleotide sequence of the flax gene LuWRI1a is shown in SEQ ID NO.1. The invention transforms the gene into Agrobacterium through genetic engineering means, infects the hypocotyl of flax, and then cultures the flax. The obtained transgenic strain grows well under salt stress and drought stress. It is found that the gene has the function of improving the salt and drought resistance of flax, and provides a theoretical basis for the construction of stress-resistant flax strains.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and more particularly to a flax gene LuWRI1a and its application in improving the salt and drought tolerance of plants. Background Art

[0002] Flax (Linum usitatissimum L.) is an annual diploid self-pollinating plant belonging to the genus Linum in the family Linaceae. Flax is a very important oil crop in the world, mainly grown in India, Canada and China. Flax seeds generally contain 40% to 50% oil, 73% polyunsaturated fatty acids, of which α-linolenic acid (C18:3, ALA) content is as high as 50%, and is used as a food raw material with high nutritional value. WRINKLED1 was originally discovered as a transcription factor in the wri1-1 mutant of Arabidopsis thaliana. Because the mutant has wrinkled epidermis, it was named WRINKLED1.

[0003] Studies have found that overexpression of WRI homologous genes in plants such as soybean, corn and rapeseed can significantly increase the seed oil content of transgenic plants. In our previous studies, we cloned two WRINKLED1 genes, LuWRI1a and LuWRI1b, from flax. We found that overexpression of LuWRI1a can increase the oil content of transgenic flax seeds without affecting the quality of flaxseed oil or seed yield. Although the various roles of WRI1 genes in different plants have been confirmed, there are few reports on whether WRI1 genes can improve plant stress tolerance.

[0004] Therefore, developing new functions of the WRI1 gene and applying it to plant salt and drought stress tolerance is a technical problem that those skilled in the art urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides an application of the flax gene LuWRI1a in improving the salt and drought resistance of plants. The LuWRI1a gene of flax is overexpressed and introduced into flax explants by Agrobacterium transformation, and three T3 homozygous strains with high LuWRI1a expression levels are obtained. The salt and drought resistance of transgenic flax are analyzed by simulating salt treatment and drought treatment using NaCl and PEG-6000. The function of the LuWRI1a gene in flax stress resistance is studied by observing the plant phenotype of transgenic flax, measuring relevant physiological indicators, and analyzing the expression of stress response genes in transgenic flax, so as to provide a theoretical basis and germplasm resources for breeding new varieties of drought-resistant and salt-alkali-resistant flax.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The flax gene LuWRI1a is characterized in that the nucleotide sequence of the flax gene LuWRI1a is shown as SEQ ID NO.1.

[0008]

[0009] As the same inventive concept as the above technical solution, the present invention also requests protection for the protein encoded by the flax gene LuWRI1a, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] MKSPPSNDKSTTKNNKRQRKIISPSSSSSSLSSLTSNSSCSSNSSNNSSPPSPSCSCSSSPCSSCDPSAVIVHPPPPPPPNHEEKPAAPPKAAKRRRRTQSSKKLTSNNNGGDHPPKSNDEDKID DPLPPPSAQGKRSSAFRGVTRHRWTGRFEAHLWDKSSWNNMHNKKGRQVYLGAYDEEEAAARTYDLAALKYWGSATTLNFPVEGYEKEMEEMSKVSREEYLASLRRRSSGFSRGVSKYRGVARHH HNGRWEARIGRVLGNKYLYLGTFNTQEEAAEAYDMAALEYRGANAVTNFDVANYVDRLKSKGDQFLQPISDVAAAEVAPEDDQEAAELYINDNAAKLEPLPLPSSSSSIEQEAAEVAMMMDMPL PPEMPPTTTTTTNNGGSMMELLELENDGNWSFCFEYPEENNAASSQLASEEGCCILPELFDVDGGGFQLEDIDYLVFDSYPPPVAVDDDDVGKSVKEKLLSEEDLSRSPSCSTTTSVSCN, such as SEQ Shown as ID NO.2.

[0011] As an inventive concept identical to the above technical solution, the present invention also requests protection for the use of the flax gene LuWRI1a in constructing biomaterials, wherein the biomaterials include recombinant vectors and recombinant strains.

[0012] Preferably, the recombinant vector is pE101-LuWRI1a.

[0013] As the same inventive concept as the above technical solution, the present invention also requests protection for the application of the flax gene LuWRI1a in improving the salt and drought tolerance of flax.

[0014] Preferably, the flax gene LuWRI1a upregulates the expression of LuAREB2, LuDREB2, LuLEA and LuNCED genes, and regulates the stress tolerance of the flax transgenic plants.

[0015] Preferably, the flax gene LuWRI1a enhances the activity of antioxidant enzymes in flax under salt stress and drought stress and reduces oxidative damage to flax cell membranes.

[0016] As the same inventive concept as the above technical solution, the present invention also requests to protect the application of the flax gene LuWRI1a in constructing salt- and drought-resistant flax strains and molecular breeding.

[0017] Preferably, the construction process of the flax strain includes: constructing a recombinant vector pE101-LuWRI1a with the flax gene LuWRI1a, transforming the recombinant vector pE101-LuWRI1a into Agrobacterium to obtain a recombinant strain, and finally infecting flax with the recombinant strain to obtain a flax strain resistant to salt stress and drought stress.

[0018] As the same inventive concept as the above technical solution, the present invention also claims protection for a flax strain that is tolerant to salt and drought stress, which is constructed by the method described.

[0019] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses the discovery of the flax gene LuWRI1a and identifies the function of the gene, providing a theoretical basis for the construction and molecular breeding of flax stress-resistant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The attached figure shows the process of obtaining transgenic flax; A: Agrobacterium infects flax hypocotyls; B: Hypocotyls are induced to form adventitious buds; C: Rooting; D: Transplantation of transformed seedlings;

[0022] Figure 2 The attached figure is a diagram showing the molecular identification results of transgenic flax; A: PCR detection of LuWRI1a transgenic flax, M: Marker DL2000; W: blank control; 1-24: transgenic flax; B: expression analysis of transgenic flax LuWRI1a; WT: wild type control (flax cultivar Longya No. 10); OE-2, OE-20, OE-22: overexpression flax lines;

[0023] Figure 3 The attached figure shows the phenotypes of wild-type plants and transgenic plants after stress treatment;

[0024] Figure 4The attached figure shows the analysis of transgenic plant traits under salt stress and drought stress; A: plant height; B: taproot length; C: lateral root number; D: leaf number;

[0025] Figure 5 The attached figure is a diagram showing the enzyme activity assay of flax overexpressing LuWRI1a;

[0026] Figure 6 The attached figure is an expression analysis diagram of stress-related genes in transgenic flax under stress treatment. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Test materials used in the examples

[0029] The test materials were the flax variety (Linum usitatissimum) Longya No. 10 and the LuWRI1a overexpression T3 homozygous transgenic line LuWRI1a-OX-X, and the seeds were provided by the Flax Research Laboratory of the Crop Research Institute of Gansu Academy of Agricultural Sciences. The well-growing transgenic lines were selected for PCR identification of positive lines.

[0030] Example 1 Construction of LuWRI1a overexpressing flax strains

[0031] The flax variety Longya 10 was transformed by Agrobacterium-mediated method. The hypocotyls of flax were infected with Agrobacterium GV3101 containing the recombinant plasmid pE101-LuW RI1a. Figure 1 ; Cultivate in culture medium, change the culture medium every 2 weeks until complete plants are obtained. At the same time, non-transgenic "Longya No. 10" (wild type) is used as a negative control. Select well-growing regenerated plants for PCR identification of positive strains. Through PCR identification and screening of positive plants, 5 homozygous transgenic flax strains were obtained ( Figure 2 A). According to the results of qRT-PCR, three different transgenic lines with higher expression levels, OE-1, OE-2 and OE-5, were selected for the next test ( Figure 2 B), harvest T1 generation seeds by greenhouse cultivation, culture T1 generation plants in pots in the greenhouse, and obtain T2 generation seeds by screening and identifying positive plants.

[0032] The primer sequences are shown in SEQ ID NO.19 to SEQ ID NO.20.

[0033] F: 5′-CCACATGAAATCGCCGCCGTCAAACGAC-3′, as shown in SEQ ID NO. 19;

[0034] R: 5′-CAAGAAACCGATGTTGTTGTTGA-3′, as shown in SEQ ID NO.20.

[0035] Example 2 Comparison of growth status between transgenic strains and wild-type strains under adverse stress

[0036] The potting method was used. The pot was 18 cm high and 15 cm in diameter. Imported Danish quality peat soil and vermiculite were mixed evenly in a volume ratio of 3:1. An appropriate amount of mixed nutrient soil was put into a small flower pot. About 15 flax seeds with full grains and uniform size were selected (T3 generation homozygous seeds, the control group was wild type), sown in the flower pot, and placed at room temperature. 1 / 2 Hogland nutrient solution was used for cultivation for 1 month, and 100 mL / time was irrigated every 3 days. The light intensity was 130 μmol·m -2 ·s -1 , the photoperiod was 16h / 8h (light / dark), and the plants were cultured at room temperature for 4 weeks for stress treatment. The experiment was set up with 4 treatments and 3 replicates. Seedlings with the same growth potential were selected for the following treatments:

[0037] (1) Salt stress treatment: 1 / 2 Hoagland nutrient solution was used to prepare 200 mmol / L -1 NaCl nutrient solution, 100 mL every 3 days;

[0038] (2) PEG-6000 simulated drought treatment: 25% PEG nutrient solution was prepared with 1 / 2 Hoagland nutrient solution, and 100 mL was irrigated every 3 days;

[0039] After 2 weeks of stress treatment, the phenotypes were observed and related physiological indicators were measured. Five plants were selected from each strain to measure plant height, root length, number of lateral roots and number of leaves. Figure 3 and Figure 4 Under normal culture conditions, there were no significant differences in plant height, taproot length, lateral root number, and leaf number between the transgenic lines and the wild type. -1 Under NaCl stress, the wild-type flax plant height was lower, the leaves turned yellow, and they were severely dried up. The degree of wilting was more serious than that of the transgenic line, which indicated that the flax overexpressing the LuWRI1a gene had a stronger salt tolerance than the wild-type flax. After PEG-6000 stress treatment, neither the wild-type plants nor the transgenic lines showed wilting, and the leaves of the transgenic lines partially lost their green color, with no significant phenotype difference ( Figure 3). The root growth of wild-type plants was inhibited, the number of lateral roots was significantly reduced, and the average number of lateral roots of transgenic plants was significantly different from that of wild-type plants ( Figure 4 ), increased by 36.36%, 30.91% and 23.63% respectively. The average root length increased by 9.46%, 15.44% and 8.63% respectively, and the average leaf number increased by 18.07%, 31.73% and 9.64% respectively; after two weeks of drought stress treatment, there was no significant difference between the transgenic plants and the control, the average taproot length increased by 3.08 times, 5.17 times and minus 0.23 times respectively, the average lateral root number increased by 25.9%, 22.29% and 22.89% respectively, and the average leaf number increased by 5.5 times, 6.88 times and 4.59 times respectively. After salt stress and drought stress treatment, the relative plant height, taproot length, lateral root number and leaf number of the transgenic plants increased, and after salt stress treatment, the various indicators of the transgenic plants were significantly higher than those of the control, indicating that the transgenic lines overexpressing LuWRI1a have higher tolerance to salt stress.

[0040] In addition, 5 flax plants were randomly selected from each group of different treatment materials, and root tissues and aboveground tissues were cut, with 3 replicates for each plant line. After quick freezing in liquid nitrogen, they were stored in a -80℃ refrigerator.

[0041] Example 3 Comparison of transgenic strains and wild-type strains in SOD activity, CAT activity, ascorbate peroxidase (APX) activity and malondialdehyde (MDA) content

[0042] The activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and malondialdehyde (MDA) are important physiological indicators of plant stress resistance. Under normal conditions, the APX activity of transgenic plants was higher than that of wild-type plants, and there was no significant difference in CAT activity, SOD activity and MDA content. After salt stress and drought stress, the activities of the three antioxidant enzymes were significantly higher than those of the control, while the MDA content was lower than that of the control.

[0043] After salt stress treatment, the SOD activity of wild flax was 109.73 U·g -1 The SOD enzyme activities of transgenic flax were 134.90, 138.83 and 129.11 U·g -1 , which was 0.23-, 0.27- and 0.18-fold higher than that of the wild type ( Figure 5 A); the CAT enzyme activity of wild flax is 36.80 U·g -1 The CAT enzyme activities of transgenic flax were 45.30, 53.38 and 42.32 U·g -1 , which was 0.23-, 0.45- and 0.15-fold higher than that of the wild type ( Figure 5 B); The APX enzyme activity of wild-type flax was 29.07 U·g -1 The APX enzyme activities of transgenic flax were 41.85, 41.91 and 40.67 U·g -1 , which was 0.44, 0.44 and 0.40 times higher than that of the wild type ( Figure 5 C); the MDA content of wild-type flax was 75.50 nmol·g-1, and the MDA content of transgenic flax was 66.33, 60.79 and 63.0 nmol·g-1, respectively. -1 , which were 0.12, 0.19 and 0.17 times lower than those of wild-type flax ( Figure 5 D).

[0044] After drought stress treatment, the SOD activity of wild flax was 109.71 U·g -1 The SOD enzyme activities of transgenic flax were 134.40, 146.28 and 122.86 U·g -1 , which was 0.22-, 0.33- and 0.12-fold higher than that of the wild type ( Figure 5 A); the CAT enzyme activity of wild flax is 36.22 U·g -1 The CAT enzyme activities of transgenic flax were 39.76, 37.81 and 43.53 U·g -1 , which was 0.10-, 0.04- and 0.20-fold higher than that of the wild type ( Figure 5 B); The APX enzyme activity of wild-type flax was 32.24 U·g -1 The APX enzyme activities of transgenic flax were 41.96, 42.59 and 38.59 U·g -1 , which was 0.30-, 0.32- and 0.20-fold higher than that of the wild type ( Figure 5 C); the MDA content of wild flax is 71.85 nmol g -1 The MDA contents of transgenic flax were 39.15, 40.18 and 40.38 nmol·g -1 , 0.46-fold, 0.44-fold and 0.44-fold lower than those of wild-type flax ( Figure 5 D). The results showed that overexpression of LuWRI1a could enhance the activities of antioxidant enzymes and alleviate the oxidative damage to flax cell membranes under salt and drought stress.

[0045] Example 4 Analysis of the expression of stress response genes in transgenic flax

[0046] In order to investigate the possible molecular mechanism of LuWRI1a in response to adverse stress, the expression levels of four abiotic stress-responsive genes, LuAREB2 (ABA-responsive element binding), LuDREB2 (dehydration-responsive element binding), LuLEA (late embryogenesis-abundant protein) and LuNCED (9-cis-epoxycarotenoid dioxygenase), in wild-type flax and transgenic flax were analyzed.

[0047] Primer synthesis

[0048] Based on the genome sequence of Linum usitatissimum 10 published on NCBI, Primer5.0 was used to design fluorescence quantitative primers. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene. All primers were synthesized by Shanghai Biochemical Company (Table 1).

[0049] Table 1 Real-time fluorescence quantitative PCR primers

[0050]

[0051] Real-time fluorescence quantitative (qRT-PCR) analysis showed that under salt stress and drought stress, stress-related genes were upregulated ( Figure 6 ). The expression level of LuAREB2 in transgenic plants was significantly higher than that in wild type plants under normal and adverse stress conditions ( Figure 6 A). Under normal conditions, there was no significant difference in the expression of LuDREB2 between wild-type and transgenic plants. After salt stress and drought stress treatment, the gene expression was significantly upregulated compared with the control ( Figure 6 B). The expression level of LuLEA5 in transgenic plants was significantly higher than that in wild type under normal conditions and salt stress treatment, but there was no significant difference among strains after drought stress treatment ( Figure 6 C). Under normal conditions, LuNCED had no significant difference compared with the control. After salt stress and drought stress treatment, the expression of the gene was slightly upregulated compared with the control, but the difference between strains was not significant ( Figure 6 D) The results showed that under stress, especially salt stress, LuWRI1a in transgenic plants participated in regulating the stress tolerance of flax transgenic plants by upregulating the expression of stress response genes such as LuAREB2, LuDREB2, LuLEA and LuNCED.

[0052] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0053] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Flax Gene LuWRI1a Application in improving salt and drought tolerance of flax, characterized in that: The flax gene LuWRI1a The nucleotide sequence of the flax gene is shown in SEQ ID NO.

1. LuWRI1a The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: Flax gene LuWRI1a Increase LuAREB2、LuDREB2、 LuLEA and LuNCED Gene expression regulates the stress tolerance of flax transgenic plants.

3. The use according to claim 1, characterized in that: Flax gene LuWRI1a Enhance the activity of antioxidant enzymes in flax under salt stress and drought stress and reduce the oxidative damage to flax cell membranes.

4. Flax Gene LuWRI1a The application of the method in constructing salt stress-tolerant and drought stress-tolerant flax strains and molecular breeding is characterized in that: The flax gene LuWRI1a The nucleotide sequence of the flax gene is shown in SEQ ID NO.

1. LuWRI1a The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

5. The use according to claim 4, characterized in that: The process of constructing the flax strain includes: using the flax gene LuWRI1a Construction of recombinant vector pE101- LuWRI1a , the recombinant vector pE101- LuWRI1a The recombinant strain was transformed into Agrobacterium to obtain a recombinant strain, and finally the recombinant strain was used to infect flax to obtain a flax strain that is resistant to salt stress and drought stress.

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