CN112680446A

CN112680446A - Corn heat-inducible promoter ZmBI-1-pro and application thereof

Info

Publication number: CN112680446A
Application number: CN202011597829.6A
Authority: CN
Inventors: 刘琳; 刘艳春
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-04-20

Abstract

The invention relates to a corn heat-inducible promoter ZmBI-1-pro and application thereof, wherein the nucleotide sequence of the promoter ZmBI-1-pro is a nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence which is complementary with the nucleotide sequence shown in SEQ ID NO.1, or a DNA molecule which has homology of more than 75% with the nucleotide sequence shown in SEQ ID NO.1 and also has a starting function. The corn heat-inducible promoter ZmBI-1-pro can start the expression of heat regulation related target genes, has a certain guiding function for cultivating heat-resistant plant varieties, can be applied to plant genetic improvement, and has important significance for heat regulation research.

Description

Corn heat-inducible promoter ZmBI-1-pro and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a corn heat-inducible promoter ZmBI-1-pro and application thereof.

Background

With the progress of social science and technology and the acceleration of industrialization process, global climate is warmed, extreme weather such as high-temperature weather frequently occurs, and the distribution range is further expanded, thus seriously affecting the production and food safety of crops.

Corn is one of the grain crops with the widest planting area in the world, and the corn seeding area and the total yield of China are the second place in the world. Although corn is a temperature-favored crop, a series of physiological and biochemical reactions occur in the crop body under the condition of abnormal high temperature, and finally the plant morphology is changed and the yield is reduced. The most important period in the life of corn is the period of joint growth of vegetative growth and reproductive growth, and is also the most vigorous period in growth and development, the vegetative growth is the basis of plant growth and development, and if high temperature is met at the moment, the plants can be dysplastic and even stop developing, thus having serious influence on the growth in the later period. The sensitivity of corn to high temperature varies in different growth and development stages. The physiological and biochemical reactions inside the body can be expressed in the morphological characteristics outside the body.

The plant can ensure normal growth and development of the plant in an external environment by regulating the ordered expression of different genes in time and space. Eukaryotes have a variety of ways to regulate gene expression and regulation. Eukaryotic genes are regulated from DNA, transcription, post-transcriptional processing, translation, and post-translational processing levels. Among them, the regulation of higher plant genes is mainly performed at the transcriptional level.

Promoters are mainly classified into constitutive promoters, tissue-specific promoters and inducible promoters. Inducible promoters initiate transcription of a foreign gene only upon stimulation by certain physical or chemical signals or upon the development of a plant to a particular stage. The promoter not only can accumulate the expression product of the target gene in a certain space and time, increase the regional expression quantity and improve the stress resistance of the plant, but also can avoid the negative effect of the plant development caused by the over-expression of the exogenous gene initiated by a constitutive promoter, can solve the problems of gene silencing and food safety in transgenic application to a certain extent, and is an ideal promoter for carrying out genetic engineering breeding by applying a plant transgenic technology. The inducible promoter has very low starting activity even does not start downstream transcription under the normal adaptive growth condition of the plant, but can efficiently start transcription when being stressed by the outside. Inducible promoters are therefore essential for plants to adapt better to the natural environment and to maintain normal growth. Wherein, the heat-induced promoter can induce a large amount of heat-resistant related genes to be expressed in a short time when the external temperature is higher, so as to adapt to a high-temperature environment. Therefore, the promoter is used as an important regulation and control element on the transcription level, and has important application value in the field of genetic engineering for developing and utilizing efficient and specific promoters.

Disclosure of Invention

The invention aims to provide a corn heat-inducible promoter ZmBI-1-pro and application thereof, and solves the problem of abnormal corn development caused by a high-temperature environment in the prior art.

The inventors of the present invention have studied on heat resistance of plants and found a promoter that drives expression of a foreign gene under high-temperature induction conditions.

The invention aims to provide a heat-inducible promoter, which can ensure that an exogenous target gene is specifically expressed only under heat induction, has no adverse effect on the growth and development of plants at proper temperature and has very important application potential. Reference herein to "plant" is to a monocotyledonous plant, such as maize, wheat, sorghum, preferably maize.

The technical scheme adopted by the invention for solving the technical problem is as follows: a corn heat-inducible promoter ZmBI-1-pro has a nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.1, or a DNA molecule which has more than 75% homology with the nucleotide sequence shown in SEQ ID NO.1 and also has a starting function.

Furthermore, the nucleotide sequence of the corn heat inducible promoter ZmBI-1-pro is the nucleotide sequence shown in SEQ ID NO.1, or the nucleotide sequence which is complementary with the nucleotide sequence shown in SEQ ID NO.1, or the DNA molecule which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and also has the starting function.

It should be noted that, all the artificially modified DNA molecules having 75% or more identity to the nucleotide sequence shown in SEQ ID NO.1 isolated by the present invention and having a priming function are derived from the nucleotide sequence of the present invention and are identical to the nucleotide sequence of the present invention.

The invention provides an application of ZmBI-1-pro host cells in plant breeding.

The invention provides an application of ZmBI-1-pro host cells in heat-resistant plant breeding.

The invention discovers and positions a new heat-induced ZmBI-1-pro for the first time. The heat-induced ZmBI-1-pro can start the expression of heat regulation related target genes, can culture heat-resistant plant varieties, can be applied to the genetic improvement of plants, and has important significance on the research of heat regulation.

The corn heat-inducible promoter ZmBI-1-pro and the application thereof have the following beneficial effects: the corn heat-inducible promoter ZmBI-1-pro can specifically drive exogenous genes to express in different tissues of corn under the high-temperature induction condition, such as leaves, stems, roots, tassels, female ears and the like, so that the corn heat-inducible promoter ZmBI-1-pro can be used for improving and improving the growth characteristics of plants, particularly the high-temperature resistance of plants, and further effectively improving the high-temperature resistance of the plants.

Drawings

FIG. 1 is a comparison graph of RNAseq expression values in maize leaf tissues treated with different high temperature times by a maize heat inducible promoter ZmBI-1-pro driving BI gene of the invention;

FIG. 2 is a comparison graph of RNAseq expression values in corn stem tissues treated with different high temperature times by the corn heat-inducible promoter ZmBI-1-pro driving BI gene of the invention;

FIG. 3 is a comparison graph of RNAseq expression values in corn root tissues treated with different high temperature times by the corn heat-inducible promoter ZmBI-1-pro driving BI gene of the invention;

FIG. 4 is a histogram of fluorescent quantitative PCR gene expression in leaf tissue of ZmBI-1-pro promoter driving BI gene after corn has been treated at 38 ℃ for 0h, 2h, 12h and 24 h;

FIG. 5 is a histogram of fluorescent quantitative PCR gene expression in stem tissue of ZmBI-1-pro promoter driving BI gene after corn has been treated at 38 ℃ for 0h, 2h, 12h, 24 h;

FIG. 6 is a histogram of fluorescent quantitative PCR gene expression in root tissue of ZmBI-1-pro promoter driving BI gene after corn has been treated at 38 ℃ for 0h, 2h, 12h, 24 h;

FIG. 7 is a map of GUS, which contains the promoter ZmBI-1-pro recombinant vector pCAMBIA 3301-ZmBI-1-pro.

Detailed Description

The maize heat-inducible promoter ZmBI-1-pro and its use according to the invention are further illustrated below with reference to the accompanying drawings and examples:

the invention discovers and positions a new heat-induced ZmBI-1-pro promoter for the first time. The heat-induced ZmBI-1-pro promoter can start the expression of heat regulation related target genes, can culture heat-resistant plant varieties, can be applied to the genetic improvement of plants, and has important significance on the research of heat regulation.

The invention respectively collects 3 different tissue samples, each group has 3 biological repetitions, extracts RNA in the samples and then carries out RNA high-throughput sequencing analysis by respectively carrying out heat treatment on corn plants in a corn V6 stage (a corn V6 stage is a corn sixth leaf is completely unfolded, and a tassel growth cone begins to extend, namely a corn stalk is pulled out, and the corn plants are respectively subjected to heat treatment for 0, 2, 24 and 48 hours.

The present invention relates to RNA Seq high-throughput sequencing, analyzing root-specific expressed genes, see table 1 and fig. 7.

TABLE 1 plantarCARE prediction of regulatory elements in the ZmBI-1 promoter sequence

Example 1

1 Material

1.1 plant Material

The maize variety with different light treatment times was B73.

2 method

2.1 extraction of RNA from the plants after thermal Induction

The corn is cultivated to a period of V6 (a period of V6 is that the sixth leaf is completely unfolded, and the male ear growth cone begins to extend, namely the node is pulled), illumination treatment is carried out for 0h, 2h, 24h and 48h at the same time, the leaves, stems and roots in a period of V6 are respectively taken to carry out RNA extraction by a TRIzol method, and the obtained product is sent to a company to carry out RNA Seq library construction work and high-throughput sequencing.

2.2 promoter information analysis

Analysis of cis-acting elements in the heat-inducible expression promoter was performed by on-line analysis of the promoter software plantacare (bioinformatics. psb. element. be/western tools/plantacare/html /). Among them, G-box is found in many plant promoters, and it is an essential element for the function of many stress-responsive promoters, and plays an important role in the process of the plant promoters affecting light, anaerobic environment and plant hormones.

2.3 results

After the corn at the V6 stage is subjected to heat treatment for 0h, 2h, 24h and 48h, the gene expression level is the highest in the corn leaves subjected to heat treatment for 2h as shown in FIG. 1; FIG. 2 shows that the gene expression level is highest in the plants subjected to heat treatment for 2 hours in corn stalks; FIG. 3 shows that the gene expression level was higher in roots subjected to heat treatment for 2 hours than in other treatment times. FIGS. 4, 5 and 6 show that the promoter was also heat-induced in the leaf, tassel and female ear in the same manner as in the case of the maize at the time R1, indicating that the promoter of the gene is a heat-inducible promoter.

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings, and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Sequence listing

<110> Shenzhen university

<120> corn heat-inducible promoter ZmBI-1-pro and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2000

<212> DNA

<213> maize (Zea mays Linn. Sp.)

<400> 1

tgactataac aatcgatgca catgatacat aaactagtta ttataatgta attcaaaaca 60

ttcttgagtt taattttgta ggaacaaaac tctaaaagtg gtattctttg tgtgtgattg 120

gttcgatacc tgtaatgtga ttagacaaaa tcaatatggc ataactgaac tcaagcacaa 180

ggaacgactt cgaggccata acaattttat cgttgcacac cactgcgagc agatgtacta 240

tatatcatac ccaagcccaa agttagaagc atggtgggta gttcacaagg taaaccctag 300

gaaactttta catactactt gcattgctgg ttattgattt gaagacgact aggtcgatgc 360

gttttgccaa aaagaagaat tgctaacttc ttttgttgac gatgagggtg ttgcacccaa 420

ctcactagtc ggagaacgag acgatatcta tactttctac tatattttaa tacattccct 480

taaaagattt caccattaca ttcaatatat ctcctaaaat cccccccccc ccacctatct 540

ctcctatatg aactaccaca tatatttaat tgtttttttt ccaacagttg gggtgtgcct 600

gctacatcac ctggcattgg cattttatca aacaccacgc ctgcacaccg cggctagcaa 660

gccggtgctc gccttcgatg tgtactgctc atcttctcca gtgcgcgtgc cgtgcagacg 720

ctcgctgggc gtagagagca gctcaccgca ctcgctgcgg aggccgaccg ccgagctcag 780

ctagctcctt atatgcggtt gatacagtgc tctccctccc tatatgcggt ttgaggacgt 840

ttctacgcat ctgttagaga gagagagccc agtgccgagt tggtgtcggt gctgcatgtc 900

ccttcctgac gtttgctgga gcccggaagt catgtatgaa aaaattaaaa ttggcatgtg 960

aagccagtaa aaggtgaaaa ctccttaaca tgaaactata caaccattta cgactatctc 1020

tataatgtat tcatttttat atctatatct aaactctatt ataaaaaaca gtattttagg 1080

tgattatacg gataaactga agcacggtat aagagccacg ggcacattga cgatgataac 1140

ggttcccaga tattaccgta tctactgttg cttatgtttt gctttgccaa gatagatccc 1200

gataccgtac ccgcacgggt aattttacct gttggataaa ccatacccat tagaaaagtc 1260

ttaaattcca atatatcaat cactcaaaat attaaataaa cataaaaaat aagtttaact 1320

ttacatataa caaatgatat gattacataa cttgttagct agataaataa tagctagaga 1380

aatgttttat tttagctaag atgaattcta ttagatggtt ataatggtat tgatacgagt 1440

atattgtaca tataggtaga cgggtatgtg tagtaccaac ccatatccgt ctatcaatgg 1500

gtagatgttc ttatttatta aaatatccac gagtaaaaaa acattccgtg tccgctttta 1560

tatcgaataa aaccaaccga gtagtcagat tttagatacc cattattatt cccaagagag 1620

accccgataa attaggctgg acaaactaga aagcacgggc gagccacaga agaaaacgca 1680

tgccagttgc ctgttggctg ttggcctgtt gcgggcttgc ggagataccc cggcctgcta 1740

agcaaaaata aaagcaatgg gggtatcact tccgagttcc ggccccgttc tctactctcc 1800

tcccttcaag aaatcagaca gagtccacca gacagacaga cagacacgcg attagaatgc 1860

cgtccgcgca ccagtgcccc acgtgtcagc ctccggtccc cgccacgtcg gaccacccgc 1920

accgccgaag ggacggaatc ctcgcgaacc ttcactccct ctctcgcgcc atcctgcgct 1980

ctccggcatt gggttggccc 2000

Claims

1. A corn heat-inducible promoter ZmBI-1-pro is characterized in that the nucleotide sequence is a nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence which is complementary with the nucleotide sequence shown in SEQ ID NO.1, or a DNA molecule which has homology of more than 75% with the nucleotide sequence shown in SEQ ID NO.1 and also has a starting function.

2. A corn heat-inducible promoter ZmBI-1-pro is characterized in that the nucleotide sequence is a nucleotide sequence shown in SEQ ID NO.1, or a nucleotide sequence which is complementary with the nucleotide sequence shown in SEQ ID NO.1, or a DNA molecule which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and also has a starting function.

3. A recombinant vector comprising the promoter pCAMBIA3301-ZmBI-1-pro of claim 1 or 2 GUS.

4. Use of the promoter ZmBI-1-pro according to claim 1 or 2 for plant breeding after induction by heat.

5. Use of the promoter ZmBI-1-pro according to claim 1 or 2 in breeding of heat-tolerant monocot plants.