CN113564176B

CN113564176B - Wheat TaHAL3-7A gene and application thereof in regulating drought resistance of crops

Info

Publication number: CN113564176B
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: 2023-06-16
Anticipated expiration: 2041-06-07
Also published as: CN113564176A

Abstract

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

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 drought resistance of crops.

Background

Wheat (Triticum aestivum L.) is an important food crop in the world, is highly adaptable and widely distributed, and provides about 20% of the food calories to humans (Kulkarni et al, 2017; mao et al, 2020). As global climate warming and water resource shortage become increasingly more problematic, drought has become one of the major factors affecting crop growth and yield (salam et al, 2019). Therefore, the excavation of excellent drought-resistant genes and the use thereof in breeding will have great significance for the safety and sustainable development of future foodstuffs (Kulkarni et al, 2017). At present, research on drought resistance of plants is carried out by utilizing a genetic engineering technology, a large number of related genes are cloned, and the genes are transferred into plants for drought resistance mechanism research. Some experiments show that the drought resistance of transgenic plants can be improved by transferring genes related to drought resistance in the plants and other organisms into the plants and transcription and translation products of the genes.

HAL3 is a highly conserved flavoprotein found in fungi, plants and animals and plays an important role in cell cycle switching and salt tolerance (Sun et al 2009). HAL3 proteins have been reported to improve salt tolerance in yeast, arabidopsis, tobacco, rice, soybean, apple, and the like (Ana et al, 1999; ikuko et al, 2004; guo et al, 2009; sun et al, 2009; yang et al, 2020). However, no research report on the wheat TaHAL3 gene has been found.

Disclosure of Invention

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

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

a wheat TaHAL3-7A gene has a CDS sequence shown in SEQ ID NO.1, and is specifically as follows: ATGGCTACATCAGAACCGGTACAGGAAAGCTGGGAGCTGGAACCCAGTAGGCCTCGGGTCCTCCTTGCTGCTTCAGGGAGTGTAGCTGCTATAAAATTCGAGAGCCTCTGTCGTGTCTTCTCCGAGTGGGCGGAAGTCCGAGCTGTGGCGACCAAGTCATCATTGCACTTTATTGAGAGATCATCTCTGCCTAGCGACGTCATTCTTTACACTGATGATGATGAGTGGTCTACCTGGACGAAGATAGGAGACGAGGTTCTGCACATAGAGCTGCGGAAGTGGGCAGACATCATGGTGATCGCCCCCTTATCAGCAAACACTCTGGCCAAGATCGCTGGTGGGTTATGCGACAACCTCCTGACATGCATAGTGCGGGCGTGGGACTACAAGAAGCCGATCTTCGCCGCTCCAGCCATGAACACCTTCATGTGGAACAACCCGTTCACGGCGCGCCACATCGAGACGATCAACCAACTCGGGATTTCCTTGGTCCCACCCACCACGAAGAGGCTGGCCTGCGGCGACTACGGGAACGGCGCGATGGCTGAGCCTTCACAGATCCATACGACCGTGAGGCTCGCGTGCAAGTCACATACGTTCGGCACGGGCAATTCACCCGCGATCCCTTCCAGCAGCCACCCTGTCTAG

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

MATSEPVQESWELEPSRPRVLLAASGSVAAIKFESLCRVFSEWAEVRAVA

TKSSLHFIERSSLPSDVILYTDDDEWSTWTKIGDEVLHIELRKWADIMVIAP

LSANTLAKIAGGLCDNLLTCIVRAWDYKKPIFAAPAMNTFMWNNPFTARH

IETINQLGISLVPPTTKRLACGDYGNGAMAEPSQIHTTVRLACKSHTFGTG

NSPAIPSSSHPV

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

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

Drawings

FIG. 1 shows the analysis of the expression level of TaHAL3 gene in transgenic lines according to example 2 of the present invention. Wherein:

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

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

FIG. 2 is a diagram showing drought resistance identification of transgenic rice in example 3 of the present invention. Wherein:

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

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

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

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

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

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

FIGS. 3G-H are ear length and spike count investigation analyses of Fielder and Fielder-OEW, respectively, after recovery from drought treatment.

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 present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

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

Example 1

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

The wheat TaHAL3-7A gene is cloned from a wheat sequencing variety China spring by taking a rice OsHAL3 protein sequence as a reference and combining PCR amplification sequencing verification through bioinformatics analysis. Amplifying with cDNA of China spring as template, recovering target fragment, homologous recombination and connection reaction with the vector, and final colony PCR and sequencing to obtain positive cloning vector pUbi TaHAL3 and pUbi TaHAL 3. Flower 17 (ZH 17) in rice acceptor material and wheat acceptor material field are transformed by agrobacterium-mediated method.

Example 2

Analysis of expression level of TaHAL3-7A Gene in transgenic lines

And identifying transgenic rice plants and wheat plants by using TaHAL3 gene specific primers through a conventional PCR technology, and screening to obtain positive transgenic lines. Leaves of positive transgenic plants are taken, RNA is extracted by using a TaKaRa Total RNA kit, and cDNA is synthesized by reverse transcription using oligo (dT) as a primer. The relative expression level of TaHAL3 gene in transgenic rice and wheat is detected by fluorescent quantitative PCR, and three biological repeats are set for each strain. Wherein, the internal reference genes in the rice and the wheat are respectively Osubiquitin and TaGAPDH genes.

Referring to FIG. 1A, the relative expression level of TaHAL3 gene in transgenic rice is detected by fluorescence quantitative PCR, wherein the transgenic acceptor material is flower 17 (ZH 17 for short) in japonica rice variety, ZH17-OER #1, #2, #3 are three independent transgenic lines; ND, indicating undetected; referring to FIG. 1B, the relative expression of TaHAL3 gene in transgenic wheat is detected by fluorescent quantitative PCR, wherein the transgenic acceptor material is wheat variety Fielder, fielder-OEW #1, #2, #3 are three independent transgenic lines.

The results show that the expression level of TaHAL3 gene in the transgenic positive line is obviously improved relative to 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 rice OsHAL3 gene; in transgenic wheat, the expression level of TaHAL3 gene was increased by more than about 30-fold relative to the control material Fielder.

Example 3

Drought resistance identification of transgenic rice

Seeds of the rice acceptor material ZH17 and three transgenic pure lines (ZH 17-OER#1, #2, # 3) are soaked in the dark at 37 ℃ for 48 hours, transferred into deionized water after white exposure, and cultured in a climatic incubator at 28 ℃ for 16 hours under illumination/8 hours in the dark and with 50% humidity. The nutrient solution is replaced after 7 days, the rice nutrient solution containing 20% of PEG (polyethylene glycol) is treated for 7 days in the trefoil period, the survival rate is counted after 7 days of recovery of the normal nutrient solution, the survival rate (%) = (the number of survival plants after rehydration/the total number of plants before PEG stress) ×100% 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 are survival and plant height of ZH17 and ZH17-OER, respectively, after recovery from stress treatment. Significance analysis was according to the two-tailed t-test (×p < 0.01).

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

Example 4

Drought resistance identification of transgenic wheat

Seeds of wheat receptor material Fielder and three transgenic pure lines (Fielder-OEW#1, #2, # 3) were selected, germinated in a climatic incubator at 23℃with 16h light/8 h darkness and 50% humidity, and transferred to a flowerpot (containing common nutrient soil) for cultivation after the seeds were exposed to white. Stopping water supply in the three-leaf period, treating for 7 days under drought stress, adding water, recovering for 10 days, and measuring plant height and chlorophyll related index. After the heading period, the ear length and the spike number traits were investigated.

Referring to FIG. 3A, a comparison of phenotypes of Fielder and Fielder-OEW before and after drought treatment; FIGS. 3B-E are comparisons of Fielder and Fielder-OEW plant heights, chlorophyll a, chlorophyll B, and carotenoid levels, respectively, after recovery from drought treatment; FIG. 3F is a comparison of Fielder and Fielder-OEW ear phenotypes after recovery from drought treatment; FIGS. 3G-H are ear length and spike count investigation analyses of Fielder and Fielder-OEW, respectively, after recovery from drought treatment. Significance analysis was according to the two-tailed t-test (×p < 0.01).

The experimental results showed that the three wheat transgenic lines showed a significant increase in plant height compared to the control material Fielder, with higher levels of chlorophyll a, chlorophyll b and carotenoids (fig. 3A-E), and also a significant increase in spike length and spike number of the transgenic plants (fig. 3F-H). The results show that under drought conditions, the transgenic wheat of the TaHAL3 gene has stronger recovery power and the final yield property is obviously improved.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Sequence listing

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<120> wheat TaHAL3-7A gene and application thereof in regulating drought resistance of crops

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tccgagtggg cggaagtccg agctgtggcg accaagtcat cattgcactt tattgagaga 180

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atggctgagc cttcacagat ccatacgacc gtgaggctcg cgtgcaagtc acatacgttc 600

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matsepvqes welepsrprv llaasgsvaa ikfeslcrvf sewaevrava tksslhfier 60

sslpsdvily tdddewstwt kigdevlhie lrkwadimvi aplsantlak iagglcdnll 120

tcivrawdyk kpifaapamn tfmwnnpfta rhietinqlg islvppttkr lacgdygnga 180

maepsqihtt vrlackshtf gtgnspaips sshpv 215

Claims

1. The application of a wheat TaHAL3-7A gene in improving drought resistance of rice and wheat is characterized in that the drought resistance of the rice and the wheat is improved by improving the expression level of the wheat TaHAL3-7A gene, wherein the CDS sequence of the wheat TaHAL3-7A gene is shown as SEQ ID NO. 1; the protein sequence is shown as SEQ ID NO. 2.