CN112553201A

CN112553201A - Promoter ZmPSP-pro specifically expressed in maize tassel and application thereof

Info

Publication number: CN112553201A
Application number: CN202011438614.XA
Authority: CN
Inventors: 刘琳; 赵俊伊; 何娟; 莫蓓莘; 陈雪梅
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-03-26

Abstract

The invention relates to a promoter ZmPSP-pro specifically expressed in maize tassel and application thereof, wherein the nucleotide sequence of the promoter ZmPSP-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 maize tassel specific expression promoter ZmPSP-pro can start the exogenous gene to specifically express in the plant tassel, thereby avoiding the adverse effect caused by the continuous expression of the exogenous gene in other tissues of the plant, and having important significance and wide application value for the targeted character improvement of transgenic maize crops.

Description

Promoter ZmPSP-pro specifically expressed in maize tassel and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a promoter ZmPSP-pro specifically expressed by a maize tassel and application thereof.

Background

Tissue-specific promoters are expressed in a specific tissue. In the case of plants, tissue-specific promoters may initiate expression of a gene of interest in a particular tissue of a plant, such as leaves, seeds, roots, or reproductive organs. The method has important significance for the biological safety and genetic modification of transgenic crops.

It has been reported that a large number of tissue-specific promoters have been isolated, including leaf-specific promoters, stem-mass-specific promoters, root-specific promoters, flower-specific promoters, seed-specific promoters, and the like. Therefore, the search for some promoters which can more specifically regulate and control gene expression in development stages of cells, tissues, organs and the like has important significance in basic research and crop improvement under certain conditions.

Crop ear development is a complex physiological process that is synchronized with late seed development and is associated with the breakdown of the accumulation of photosynthetic products. In the embryo development process of crops, the environmental conditions such as external temperature, humidity, illumination and the like directly or indirectly influence the components and contents of amylase, endogenous hormone and other endostatin in the embryo, endosperm, pericarp and seed coat of the crops.

The development process of the ear relates to various metabolic activities, the gene expression is particularly active, and the molecular functions of the genes related to the ear development are reported to mainly relate to various aspects and pathways such as transcription factors, protein metabolism, carbohydrate metabolism, stress, signal transduction, substance transportation, cell constitution and the like. Therefore, the promoter for the ear specific expression is deeply discovered, and the promoter has important application value for pertinently improving the yield traits of the transgenic corn.

Disclosure of Invention

The invention aims to provide a promoter ZmPSP-pro specifically expressed by a maize tassel and application thereof, and solves the problems of complex and variable tassel development process, difficult control and the like in the prior art.

The technical scheme adopted by the invention for solving the technical problem is as follows: a promoter ZmPSP-pro specifically expressed by a maize tassel has a nucleotide sequence shown as SEQ ID NO. 1, or has a nucleotide sequence complementary with the nucleotide sequence shown as SEQ ID NO. 1, or is a DNA molecule which has homology of more than 75 percent with the nucleotide sequence shown as SEQ ID NO. 1 and also has a starting function.

Furthermore, the nucleotide sequence of the promoter ZmPSP-pro specifically expressed by the maize tassel 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 also provides a recombinant vector containing the promoter pCAMBIA 3301-ZmSPpro and GUS.

The invention also provides application of the promoter ZmPSP-pro in spike tissue specific expression breeding, wherein the spike tissue is an important reproductive tissue of a monocotyledon crop, preferably the spike tissue of corn; the application of the promoter ZmPSP-pro in the ear tissue specific expression breeding by promoting a target gene, in particular to the application of the promoter ZmPSP-pro in the ear tissue specific expression breeding of corn by promoting the target gene.

The invention collects samples of 6 different tissues in V6 period and R1 period (V6 period is the sixth leaf is completely unfolded, the male ear growth cone begins to extend, namely, the joint, and R1 period is the female ear filament begins to expose the bract, namely, the spinning period) of corn, and each group is set with 3 biological repetitions for RNA extraction. Spike-specific expression genes were analyzed using RNA Seq high-throughput sequencing.

The implementation of the maize tassel specific expression promoter ZmPSP-pro and the application thereof has the following beneficial effects: the promoter ZmPSP-pro specifically expressed in the maize tassel can start the plant gene to specifically express in the tassel, thereby avoiding the adverse effect caused by the continuous expression of the foreign gene in other tissues of the plant, and having important significance and wide application value for the targeted character improvement of the transgenic maize crop.

Drawings

FIG. 1 is a graph comparing the expression values of ZmPSP-pro driving PSP gene in RNA Seq in different tissues by the maize ear specific expression promoter of the invention.

FIG. 2 is a histogram of fluorescent quantitative PCR gene expression of ZmPSP-pro promoter driving PSP gene in different tissues of maize.

FIG. 3 shows a ZmPSP-pro recombinant vector containing the above-mentioned promoter

Map of GUS, pCAMBIA 3301-ZmSPpro.

Detailed Description

The promoter ZmPSP-pro specifically expressed by the maize tassel and the application thereof are further explained in the following by combining the attached drawings and examples:

TABLE 1 analysis of regulatory elements in the ZmPSP-pro promoter sequence

Example 1

1 Material

1.1 plant Material

The maize variety used in the present invention was a B73(Zea mays ssp. mays L.) cultivar B73 plant, cultivated in an artificial plant incubator at 22-26 ℃, 65% relative humidity and 14 hour photoperiod. It should be noted that the culture method is a culture method commonly used in the prior art, and detailed description thereof is omitted here.

2 method

2.1 extraction of RNA

The corn was cultivated until the period V6 and the period R1 (the period V6, namely the sixth leaf, is completely unfolded, the growth cone of the tassel begins to extend, namely, the node is pulled out; the period R1, namely, the filament of the female ear begins to expose the bract, namely, the period of silking), the leaves, stems and roots of the period V6 and the leaves, female ears and male ears of the period R1 were respectively taken, and RNA extraction was carried out by the TRIzol method, wherein samples of the above different tissues were taken, and 3 biological replicates were established for each sample. RNA Seq construction library work and high throughput sequencing were performed. As shown in FIGS. 1 to 3, the highest expression level of ZmPSP-pro gene at ear of maize at the R1 stage was obtained by the sequencing results.

It should be noted that the TRIzol method is a method commonly used in the prior art for extracting RNA, and is not described in detail here.

2.2 promoter information analysis

The cis-acting elements in the promoter were analyzed 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.

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> promoter ZmPSP-pro specifically expressed by maize tassel and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2000

<212> DNA

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

<400> 1

ggatttgctt ttgttggtcc agttgggcat tactggttag ttccctgttt tttatcacat 60

gtcgttatgt ctacctgttt acatgtatat tttctgtatg atcataggtt aatctcttac 120

tgtataacaa taaaaaaact cgtaagtcgt aaccataatc ttatgctccc agtctcagtc 180

accacacgtc acagatgaaa agcaaataca gaataacaaa cacagcaaca cactctgata 240

aaataacaaa aacataggat aatatagaag ataaaagggc aaagtgagga tgtaatccca 300

aattcccaat tctgcactgc tgttctccat tttaggtatc tacgtgttag tatgagtgac 360

agttcatggt gtcatattat cttttttgat cgtaatggtg tcatattatc aataagcact 420

tggcgactgc ttgaaaattg atatatctaa caacacatga tttgcttgaa tatactgggt 480

tatctttcaa tagagtaaat cacgatataa tattaccaac agcatgcatg acaacatgag 540

tgcacaagtg gttcacggaa ttttgccccg ctacctagtt atgtggtcag gcagttgact 600

gtccacaatg ggcaaacatc aacttataat caagagtaga agttttttgg catgtacttc 660

acagttcaca gttgtacatc cttatttgtc atttttgtgc tcagtagtcc acttcaacat 720

tgatttgatt tgcaccatgt cacatgtagt gacgatgcta tgttttggag agttagttgt 780

aaaccactgg actatgttca gctgtatgaa aacacagata aaaaaatatg atcacacggg 840

tttttgctat tggacagatg ttgatcccac aatctcatca cttactgctg caatttgttc 900

atttgcaaaa ggtaggtaag atcttgtggt acgggtgtac tgtactgccc ctctagggcc 960

caaattttct tcttaatata atgatgcaca gttctcccgc gttttcgaga aaaaaaaaga 1020

gatgaaagca gttctctcct tattttttgg agtgaaaaat acgggcgaac tattgtgaat 1080

tggactgcta tatgagatgt tgttggcagc atcaatatct gtcactaagg ctttggtttt 1140

gttgtatagg tatgaatacc tggatcgcat catccggcgg aggtttcagc ctaacacgtt 1200

caaattcgtc gcctcaaaag ttgccgcgga tggattcctc ttcggaccac tagacctcct 1260

cctgttcttc tcatatgttg gtctgggtca aggaaggagc atagagcagg tgaaggagga 1320

cgtgaagagg gacttcattc cggctctggt gttaggcgga accatctggc ctgctgtgca 1380

gatcgcgaac ttccgcttcg ttcccgtgcg gtaccagctc ctgtatgtga acttgttctg 1440

cctcctggac agctgcttcc tgtcgtggat tgagcagcag ggtgacgcct cctggaagcg 1500

gtggttcact tcgttccaga aaatcgaaga ccagaagggt aaggtttgat ttgagtcatg 1560

catggttcgt tgtctggatc agtacctgta aactccggtt gatcaaggaa gataattgcg 1620

atgccagcaa ggctgaacat tggcgtcgct atagagaatt ctttttagtc tttgacggga 1680

tgaaacggtt gccgagttgc tctgttaact gaagtgcctg tgttttgccg atgtgttaaa 1740

ctttcttttt agtctttgat ccaagatgct taagtcatgt ctgggaatgg gtggaattga 1800

cgattgcaac gcgagcaaaa gctgaatata tatcctgttg cagcagttcc agtccaggaa 1860

tagctagcga gggatgggtg gggatcatgg acggcgcaca ggcgccgacg gacgagcctg 1920

gaaacgagct ccgcctctcc tctccatcta tctatctata aagggcgcat ggcggcccct 1980

cgccgagagg agttcacgaa 2000

Claims

1. A promoter ZmPSP-pro specifically expressed in a maize tassel 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 percent with the nucleotide sequence shown in SEQ ID NO. 1 and also has a starting function.

2. A promoter ZmPSP-pro specifically expressed in a maize tassel 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-Zm PSPpro of claim 1 or 2, GUS.

4. Use of the promoter ZmPSP-pro according to claim 1 or 2 for breeding by specific expression in ear tissue.

5. The use of the promoter ZmPSP-pro according to claim 1 or 2 in maize ear tissue specific expression breeding.