CN-116790617-B - Gene mutant for reducing leaf water loss rate and application thereof

CN116790617BCN 116790617 BCN116790617 BCN 116790617BCN-116790617-B

Abstract

The invention provides a gene mutant for reducing leaf water loss rate and application thereof, and belongs to the technical field of genetic engineering. The NtMYB gene mutant for reducing the leaf water loss rate has the NtMYB gene sequence mutated as shown in SEQ ID NO. 2. The invention also provides a gene editing vector, a transformant, a kit and a method for constructing NtMYB184,184 gene mutants capable of reducing the leaf water loss rate, and application of the method. The NtMYB 184.72% leaf water loss rate of the 3835.184 gene mutant for reducing leaf water loss rate provided by the invention is lower than that of a control WT (6.87%), which shows that the NtMYB184 gene mutant (myb 184) has lower leaf water loss rate than that of the control WT.

Inventors

SONG ZHONGBANG
ZHAO LU
WANG BINGWU
SUI XUEYI
GAO YULONG
SHI JUNLI

Assignees

云南省烟草农业科学研究院

Dates

Publication Date: 20260512
Application Date: 20230116

Claims (1)

1. The application of NtMYB gene mutant is characterized in that the tobacco NtMYB184 gene is mutated into NtMYB gene mutant for reducing the water loss rate of tobacco leaves, the sequence of NtMYB184 gene is shown as SEQ ID NO.1, and the sequence of NtMYB184 gene mutant is shown as SEQ ID NO. 2.

Description

Gene mutant for reducing leaf water loss rate and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to NtMYB gene mutant capable of reducing leaf water loss rate and application thereof. Background Stomata (stomata) are holes formed by a pair of guard cells in the plant epidermis, and are channels for water removal by plant transpiration and CO 2 absorption by photosynthesis. In most terrestrial plants, stomata are opened during the day to increase photosynthesis efficiency and closed at night to minimize water loss. Drought stress affects various physiological and biochemical processes of plants, such as photosynthesis, respiration, transport system, nutrient metabolism, etc., impeding plant growth, and serious drought stress may cause death of plants. In the face of drought stress, plants produce ABA to reduce stomatal opening to reduce leaf water loss rate. Reactive Oxygen Species (ROS) levels in guard cells are key regulatory targets for stomatal movement, and a variety of exogenous stimuli and phytohormones regulate stomatal movement by regulating ROS levels. ROS act as key secondary signaling molecules that increase intracellular Ca 2+ levels, activate guard cytoplasmic membrane anion channels to release anions, closing pores. Studies show that flavonols all have antioxidant functions and can regulate ROS level in guard cells so as to influence stomatal movement. Plant flavonol synthesis is regulated by MYB transcription factors, and is cloned and obtained in Arabidopsis thaliana, grape, citrus, longspur epimedium and crabapple at present, but no related research is published in model plant tobacco, and no report that MYB influences the level of guard cell ROS by regulating flavonol synthesis so as to regulate the leaf water loss rate appears. There is a need in the art to develop a product and method for reducing the rate of water loss from a blade. Disclosure of Invention Based on the above-mentioned blank and demand in the prior art, the invention provides NtMYB gene mutant for reducing the water loss rate of leaf blades and application thereof. The technical scheme of the invention is as follows: A NtMYB gene mutant for reducing leaf water loss rate has a NtMYB gene sequence of NtMYB gene mutant for reducing leaf water loss rate, which is shown in SEQ ID NO.2, compared with NtMYB gene with a wild-type tobacco plant nucleotide sequence shown in SEQ ID NO. 1. Primers for reducing leaf loss rate and/or constructing mutants with low leaf loss rate, the upstream and downstream sequences of which are shown below, respectively: Upstream primer NtMYB-184-CP 1-F:5'-ATTGAGACCCACTTTCTCACAACA-3' Downstream primer NtMYB-184-CP 1-R:5'-AAACTGTTGTGAGAAAGTGGGTCT-3' The gene editing vector is used for reducing the leaf water loss rate and/or constructing a mutant with low leaf water loss rate, and dsDNA products obtained by annealing the vector by adopting primers with upstream and downstream sequences shown as NtMYB-184-CP 1-F and NtMYB-184-CP 1-R respectively are connected to the vector. The gene editing vector for reducing the leaf water loss rate and/or constructing the mutant with low leaf water loss rate is pHSE401,401 vector. The transformant for reducing the leaf water loss rate and/or constructing the low leaf water loss rate is a gene editing carrier for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant, which is transformed into a host cell, wherein the gene editing carrier for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant is a dsDNA product which is obtained by carrying out annealing reaction by adopting primers with upstream and downstream sequences shown as NtMYB-184-CP 1-F and NtMYB-184-CP 1-R. The transformant for reducing the leaf water loss rate and/or constructing the mutant with low leaf water loss rate is pHSE401,401 vector, and/or the host cell is selected from escherichia coli and/or agrobacterium. The kit for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant comprises the primer for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant. The kit for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant further comprises a gene editing vector for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant and/or a transformant for reducing the leaf water loss rate and/or constructing the low leaf water loss rate mutant. A method for reducing leaf water loss rate and/or constructing low leaf water loss rate mutant uses the primer with the upstream and downstream sequences shown as NtMYB-184-CP 1-F and NtMYB-184-CP 1-R to carry out gene editing on tobacco plants. The gene editing method for reducing the leaf water loss rate and/or constructing the mutant with low leaf water loss rate comprises t