CN113564176A

CN113564176A - Wheat TaHAL3-7A gene and application thereof in regulating drought resistance of crops

Info

Publication number: CN113564176A
Application number: CN202110630718.9A
Authority: CN
Inventors: 吴永振; 董嘉金; 徐彗渊; 马成学; 李晶雪; 孙晗; 秦冉; 崔法; 赵春华
Original assignee: Ludong University
Current assignee: Ludong University
Priority date: 2021-06-07
Filing date: 2021-06-07
Publication date: 2021-10-29
Anticipated expiration: 2041-06-07
Also published as: CN113564176B

Abstract

The invention relates to the field of molecular biology, and discloses a wheat TaHAL3-7A gene, wherein a CDS sequence is shown as SEQ ID NO.1, a protein sequence is shown as SEQ ID NO.2, and application of the wheat TaHAL3-7A gene in regulation and control of crop drought resistance. The invention clones TaHAL3-7A gene from wheat and constructs plant overexpression vector of TaHAL3-7A gene, and the overexpression vectors are respectively transformed into rice and wheat by agrobacterium-mediated method, thereby obviously improving the drought resistance of rice and wheat. The wheat TaHAL3-7A gene is applied to regulating and controlling the drought resistance of crops, and can provide gene resources and a method for creating new stress-resistant varieties.

Description

Wheat TaHAL3-7A gene and application thereof in regulating drought resistance of crops

Technical Field

The invention relates to the field of molecular biology, in particular to a wheat TaHAL3-7A gene and application thereof in regulating and controlling crop drought resistance.

Background

Wheat (Triticum aestivum L.) is an important food crop in the world, has strong adaptability and wide distribution range, and provides about 20% of food heat for human (Kulkarni et al, 2017; Mao et al, 2020). Due to global warming and water shortage issues, drought has become one of the major factors affecting crop growth and yield (salam et al, 2019). Therefore, the mining of excellent drought-resistant genes and the application of the genes in breeding have important significance for the safe and sustainable development of future food (Kulkarni et al, 2017). At present, great progress is made in utilizing genetic engineering technology to develop the research of plant drought resistance, a great amount of related genes are cloned and transferred into plants for the research of drought resistance mechanism. Some experiments show that genes related to drought resistance in plants and other organisms are transferred into the plants, and transcription and translation products of the genes can improve the drought resistance of the transgenic plants.

HAL3 is a highly conserved flavoprotein present in fungi, plants and animals that plays an important role in cell cycle switching and salt tolerance (Sun et al, 2009). HAL3 protein has been reported to improve salt tolerance in yeast, Arabidopsis, tobacco, rice, soybean, apple, etc. (Ana et al, 1999; Ikuko et al, 2004; Guo et al, 2009; Sun et al, 2009; Yang et al, 2020). However, no research report of wheat TaHAL3 gene is found at present.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a wheat TaHAL3-7A gene.

In order to realize the purpose, the wheat TaHAL3-7A gene provided by the invention adopts the following technical scheme:

the CDS sequence of the wheat TaHAL3-7A gene is shown as SEQ ID NO.1, and specifically comprises the following steps: ATGGCTACATCAGAACCGGTACAGGAAAGCTGGGAGCTGGAACCCAGTAG GCCTCGGGTCCTCCTTGCTGCTTCAGGGAGTGTAGCTGCTATAAAATTCGA GAGCCTCTGTCGTGTCTTCTCCGAGTGGGCGGAAGTCCGAGCTGTGGCGA CCAAGTCATCATTGCACTTTATTGAGAGATCATCTCTGCCTAGCGACGTCAT TCTTTACACTGATGATGATGAGTGGTCTACCTGGACGAAGATAGGAGACGA GGTTCTGCACATAGAGCTGCGGAAGTGGGCAGACATCATGGTGATCGCCCC CTTATCAGCAAACACTCTGGCCAAGATCGCTGGTGGGTTATGCGACAACCT CCTGACATGCATAGTGCGGGCGTGGGACTACAAGAAGCCGATCTTCGCCGC TCCAGCCATGAACACCTTCATGTGGAACAACCCGTTCACGGCGCGCCACAT CGAGACGATCAACCAACTCGGGATTTCCTTGGTCCCACCCACCACGAAGA GGCTGGCCTGCGGCGACTACGGGAACGGCGCGATGGCTGAGCCTTCACAG ATCCATACGACCGTGAGGCTCGCGTGCAAGTCACATACGTTCGGCACGGGC AATTCACCCGCGATCCCTTCCAGCAGCCACCCTGTCTAG

The protein sequence of the wheat TaHAL3-7A gene is shown in SEQ ID NO.2, and the specific steps are as follows:

MATSEPVQESWELEPSRPRVLLAASGSVAAIKFESLCRVFSEWAEVRAVA TKSSLHFIERSSLPSDVILYTDDDEWSTWTKIGDEVLHIELRKWADIMVIAP LSANTLAKIAGGLCDNLLTCIVRAWDYKKPIFAAPAMNTFMWNNPFTARH IETINQLGISLVPPTTKRLACGDYGNGAMAEPSQIHTTVRLACKSHTFGTG NSPAIPSSSHPV

the invention also aims to provide application of the wheat TaHAL3-7A in the scheme in regulating and controlling drought resistance of crops.

Compared with the prior art, the invention has the beneficial effects that: the invention clones TaHAL3-7A gene from wheat for the first time by using the existing plant genetic engineering technology, constructs plant over-expression vector of the TaHAL3-7A gene, and respectively transforms the over-expression vector into rice and wheat by an agrobacterium-mediated method, thereby obviously improving the drought resistance of the rice and the wheat. The wheat TaHAL3-7A gene is applied to regulating and controlling the drought resistance of crops, and can provide gene resources and a method for creating new stress-resistant varieties.

Drawings

FIG. 1 is an analysis of the expression level of TaHAL3 gene in a transgenic line in example 2 of the present invention. Wherein:

FIG. 1A shows the detection of the relative expression level of TaHAL3 gene in transgenic rice by fluorescence quantitative PCR, wherein the transgenic receptor material is rice japonica rice variety Zhonghua 17 (ZH17 for short), ZH17-OER #1, #2, #3 are three independent transgenic lines;

FIG. 1B shows the detection of the relative expression level of TaHAL3 gene in transgenic wheat by fluorescence quantitative PCR, wherein the transgenic acceptor material is wheat variety Fielder, and Fielder-OEW #1, #2, #3 are three independent transgenic lines.

FIG. 2 shows drought resistance identification of transgenic rice in example 3 of the present invention. Wherein:

FIG. 2A is a comparison of the phenotypes of ZH17 and ZH17-OER before and after 20% PEG treatment;

FIGS. 2B and 2C show the survival rate and plant height of ZH17 and ZH17-OER, respectively, after recovery from stress treatment.

FIG. 3 shows drought resistance identification of transgenic wheat in example 3 of the present invention. Wherein:

FIG. 3A is a comparison of the phenotypes of Fielder and Fielder-OEW before and after drought treatment;

FIGS. 3B-E are comparisons of plant height, chlorophyll a, chlorophyll B and carotenoid content of Fielder and Fielder-OEW, respectively, after recovery from drought treatment;

FIG. 3F is a comparison of Fielder and Fielder-OEW spike phenotype after recovery from drought treatment;

FIGS. 3G-H are the ear length and spikelet number survey analysis of Fielder and Fielder-OEW, respectively, after drought recovery.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The experimental procedures used in the following examples are, unless otherwise specified, conventional procedures used by those skilled in the art.

Example 1

Wheat TaHAL3-7A gene clone, vector construction and genetic transformation

The wheat TaHAL3-7A gene provided by the invention is cloned from a wheat sequencing variety Chinese spring by taking a rice OsHAL3 protein sequence as a reference and combining with PCR amplification sequencing verification through bioinformatics analysis. The cDNA of Chinese spring is used as a template for amplification, a target segment is recovered and is respectively subjected to homologous recombination and connection reaction with a vector, and a positive cloning vector is finally obtained through colony PCR and sequencing verification, wherein pUbi is TaHAL3 (OER for short, transformed rice) and pUbi is TaHAL3 (OEW for short, transformed wheat). The agrobacterium-mediated method is utilized to respectively transform the flower 17 (ZH17 for short) in the rice receptor material and the wheat receptor material Fielder.

Example 2

Analysis of expression level of TaHAL3-7A gene in transgenic line

The specific primers of the TaHAL3 gene are utilized to identify the transgenic plants of rice and wheat by the conventional PCR technology, and positive transgenic strains are obtained by screening. Taking the leaf of the positive transgenic plant, extracting RNA by using a TaKaRa Total RNA kit, and carrying out reverse transcription by using oligo (dT) as a primer to synthesize cDNA. The relative expression quantity of the TaHAL3 gene in the transgenic rice and the wheat is detected by fluorescent quantitative PCR, and three biological repeats are arranged in each strain. Wherein, the reference genes in the rice and the wheat are Osubiquitin and TaGAPDH genes respectively.

Referring to FIG. 1A, the relative expression of TaHAL3 gene in transgenic rice is detected by fluorescence quantitative PCR, wherein the transgenic receptor material is rice japonica rice variety Zhonghua 17 (ZH17 for short), ZH17-OER #1, #2, #3 are three independent transgenic lines; ND, meaning not detected; referring to FIG. 1B, the relative expression of TaHAL3 gene in transgenic wheat was detected by fluorescence quantitative PCR, wherein the transgenic acceptor material was wheat variety Fielder, and Fielder-OEW #1, #2, #3 were three independent transgenic lines.

The result shows that the expression quantity of the TaHAL3 gene in the transgenic positive strain is obviously improved compared with the expression quantity of the receptor material. Wherein in the transgenic rice, the expression level of the TaHAL3 gene is improved by more than about 200 times compared with that of the OsHAL3 gene of the rice; in transgenic wheat, the expression level of the TaHAL3 gene is improved by more than 30 times compared with that of a control material Fielder.

Example 3

Drought resistance identification of transgenic rice

The rice receptor material ZH17 and seeds of three transgenic pure lines (ZH17-OER #1, #2 and #3) are soaked in dark at 37 ℃ for 48h, and are transferred into deionized water after exposure to white, and are cultured in an artificial climate incubator with the temperature of 28 ℃, the illumination of 16 h/the darkness of 8h and the humidity of 50 percent. And (3) replacing the nutrient solution after 7 days, treating the rice in a three-leaf period for 7 days under a rice nutrient solution containing 20% of PEG (polyethylene glycol), and after the rice is replaced by a normal nutrient solution and recovered for 7 days, counting the survival rate, wherein the survival rate (%) (the number of plants surviving after rehydration/the total number of plants before PEG stress) is multiplied by 100 percent, and the plant height is measured.

See FIG. 2A for a comparison of the phenotypes of ZH17 and ZH17-OER before 20% PEG treatment and after recovery; FIGS. 2B and 2C show the survival rate and plant height of ZH17 and ZH17-OER, respectively, after recovery from stress treatment. Significance analysis was according to the two-tailed t-test (. about.p < 0.01).

The results show that the survival rate and the plant height of the three transgenic lines ZH17-OER #1, #2 and #3 are obviously increased compared with the control receptor material ZH 17.

Example 4

Drought resistance identification of transgenic wheat

Selecting wheat receptor material Fielder and seeds of three transgenic pure lines (Fielder-OEW #1, #2, #3), germinating in an artificial climate incubator with the temperature of 23 ℃, the photoperiod of 16h illumination/8 h darkness and the humidity of 50%, and transferring the seeds into a flowerpot (containing common nutrient soil) for culturing after the seeds are exposed to the white. And (3) stopping water supply in the three-leaf period, treating drought stress for 7 days, and measuring the plant height and chlorophyll related indexes after adding water and recovering for 10 days. After heading, panicle length and spikelet number traits were investigated.

See FIG. 3A for a comparison of Fielder and Fielder-OEW phenotypes before and after drought treatment; FIGS. 3B-E are comparisons of plant height, chlorophyll a, chlorophyll B and carotenoid content of Fielder and Fielder-OEW, respectively, after recovery from drought treatment; FIG. 3F is a comparison of Fielder and Fielder-OEW spike phenotype after recovery from drought treatment; FIGS. 3G-H are the ear length and spikelet number survey analysis of Fielder and Fielder-OEW, respectively, after drought recovery. Significance analysis was according to the two-tailed t-test (. about.p < 0.01).

The experimental results show that compared with the control material Fielder, the plant heights of the three wheat transgenic lines are obviously increased, the contents of chlorophyll a, chlorophyll b and carotenoid are higher (figures 3A-E), and the spike length and the number of spikelets of the transgenic plants are also obviously increased (figures 3F-H). The results show that the transgenic wheat of the TaHAL3 gene has stronger restoring force under drought conditions, and the final yield traits are also obviously improved.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

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matsvswsrr vaasgsvaak scrvswavra vatksshrss sdvytdddws twtkgdvhrk 60

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Claims

1. A wheat TaHAL3-7A gene is characterized in that a CDS sequence is shown as SEQ ID NO. 1.

2. The wheat TaHAL3-7A gene as claimed in claim 1, wherein its protein sequence is shown in SEQ ID NO. 2.

3. Use of the wheat TaHAL3-7A according to claim 1 or claim 2 for modulating drought resistance in crops.