CN113943733A

CN113943733A - Larix gmelinii endogenous efficient promoter and application

Info

Publication number: CN113943733A
Application number: CN202010619161.4A
Authority: CN
Inventors: 张勇; 郑雪莲; 张韬; 刘炳麟; 刘冠卿; 何瑶
Original assignee: University of Electronic Science and Technology of China
Current assignee: University of Electronic Science and Technology of China
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2022-01-18
Anticipated expiration: 2040-06-30
Also published as: CN113943733B

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a Larix Gmelini endogenous DNA molecule with a promoter function and application thereof. The technical problem of the invention is to provide a larch endogenous high-activity promoter element. The technical scheme for solving the technical problem is to provide a DNA molecule. The DNA molecule is shown as a) or b) or c) as follows: a) DNA molecule shown in SEQ ID No. 1; b) a DNA molecule having a homology of 99% or more, 95% or more, or 90% or more with the nucleotide sequence defined in a) and having a promoter function; c) and a DNA molecule which can be complementarily paired with the nucleotide sequence limited by a) or b) and has the function of a promoter. Experiments prove that the larch endogenous promoter disclosed by the invention can efficiently and stably drive gene expression in larches, can efficiently and stably drive gene expression in other plant species such as rice and the like, and has a good application prospect.

Description

Larix gmelinii endogenous efficient promoter and application

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a Larix Gmelini endogenous DNA molecule with a promoter function and application thereof.

Background

Larch is a type of larch belonging to the family Pinaceae (Pinaceae), the genus larch (Larix). Larch is widely distributed in mountainous areas, temperate plain areas and mountain climate areas, and there are 25 types of larch in the world. China is named as the top-ranked orchard grass in China, wherein larch (Larixprincipis-rupprechtii), dahurian larch (Larix gmelinii), Japanese larch (Larix kaempferi) and Changbai larch (Larix olgensis) are mainly distributed in northeast and northwest China; siberian larch (Larix sibirica) is mainly distributed in the Altai mountainous area of Xinjiang in northwest of China. Larch has the characteristics of early fast growth, strong stress resistance, excellent disease resistance and good ecological benefit, is an extremely important afforestation tree species in northern areas of China, and is widely applied to building protective forests and applied to returning to forest work. Because the growth of forest trees is a process with a very long period, people mainly focus on the traditional biology aspects of genetic variation research, seed source selection, wood character research, photosynthetic characteristic research and the like in the early period around the research of larch, and the genetic character improvement process of the larch is limited to a great extent.

The emergence of genome engineering technologies such as transgenic technology and genome editing brings new opportunities and challenges for the improvement of genetic traits of larch. The transgenic technology is a technology which leads the exogenous DNA with complete expression unit designed in advance by people into a receptor cell by a biotechnology means and integrates the exogenous DNA on a receptor genome, so that the exogenous gene can be stably expressed in the receptor and also has the generation genetic ability. The genome editing technology is to modify nuclease, combine or cut at a selected position of a genome after the nuclease is introduced into a receptor cell to form double-strand break, so as to realize site-specific gene knockout or expression regulation of downstream genes. Compared with the traditional crossbreeding, the transgenic technology and the genome editing technology are powerful tools for realizing the rapid directional improvement of the genetic character of the larch so as to obtain the larch species with the advantages of rapid growth, abundant yield and high quality and stress resistance.

Promoters are important functional elements for regulating the transcription and expression of downstream genes, are a piece of DNA located at the upstream of the initiation codon (ATG) of a gene, can be combined with RNA polymerase to start the transcription of the gene, and are equivalent to a 'switch' for the transcription of the gene. The promoter is a key factor for driving a target gene to express and translate in a cell, and no matter what genetic engineering technology is, the promoter needs to be applied to realize, so that the excavation of the endogenous promoter of the larch with excellent activity is a problem which needs to be solved urgently by utilizing the modern biological technology such as a transgenic technology or a genome editing technology to carry out directional genetic improvement on the larch and other researches. However, as the current larch reference genome sequence map assembly is not completed, the working progress of larch genome analysis is small, the information of endogenous promoter functional elements is less, and the mining of a promoter with excellent endogenous activity of larch is difficult.

Disclosure of Invention

The invention aims to provide a larch endogenous high-activity promoter element. The technical scheme for solving the technical problem is to provide a DNA molecule. The DNA molecule is shown as a) or b) or c) as follows:

a) DNA molecule shown in SEQ ID No. 1;

b) a DNA molecule having a homology of 99% or more, 95% or more, or 90% or more with the nucleotide sequence defined in a) and having a promoter function;

c) and a DNA molecule which can be complementarily paired with the nucleotide sequence limited by a) or b) and has the function of a promoter.

Further, the present invention provides a gene expression cassette containing the above DNA molecule or a recombinant plasmid containing the above DNA molecule.

The invention also provides a recombinant microorganism, a transgenic plant cell line or a transgenic animal cell line containing the DNA molecule.

In another aspect, the invention also provides the use of the above DNA molecule as a promoter.

Meanwhile, the invention also provides the application of the DNA molecule, the expression cassette or the recombinant plasmid in starting the expression of the target gene.

Wherein, the target gene expression is initiated in microorganisms, plant cells or animal cells.

Wherein, the plant in the application is gymnosperm or angiosperm.

Further, the gymnosperm is a plant of Pinaceae. Preferably, the plant of the Pinaceae family is a plant of the genus Larix. Further, the larch is selected from at least one of north China larch (Larix-rupprechtii), Xingan larch (Larix gmelini), Japan larch (Larix kaempferi) and Long white larch (Larix olgensis).

Wherein, the angiosperm in the application is monocotyledon. Further, the monocotyledon is a gramineae plant. Wherein the gramineous plant is a plant of the genus oryza. Further, the plants of the genus oryza are rice.

The invention also provides a method for expressing the target gene. The method uses the DNA molecule as a promoter to promote the expression of a target gene.

Wherein, the method is that the DNA molecule is operably connected to the upstream of the target gene to be expressed to start the expression of the target gene.

The invention obtains a nucleotide fragment with promoter function from larch, which is named as LarPE 004. Experiments prove that the promoter LarPE004 not only can efficiently and stably drive gene expression in larch, but also can efficiently and stably drive gene expression in other plant species such as rice and the like. The invention provides a good tool for the targeted genetic improvement of the larch by using the modern biological technologies such as a transgenic technology or a genome editing technology and the like, and has great value in the biological research of the larch.

Drawings

FIG. 1 is a schematic diagram of a skeleton vector and an expression vector used in an experiment of excavation and expression detection of a larch promoter.

FIG. 2 shows the transient transformation of green fluorescent protein expression in Larix Gmelini protoplast (Objective lens 10 ×).

FIG. 3 shows the expression of green fluorescent protein after transient transformation of rice protoplasts (Objective lens 10X).

FIG. 4 shows the expression of green fluorescent protein in rice callus (scale bar 1 mm).

FIG. 5 shows the histochemical staining of GUS in the roots of transgenic rice (scale bar 1 cm).

As the promoter is an important functional element for regulating the transcription and expression of genes, the identification of the promoter with excellent endogenous activity of the larch creates great value for the work of larch new variety cultivation, transgenic research and the like. However, the high-quality reference genome sequence of the larch is not completed at present, and difficulty is caused to the mining work of the endogenous promoter of the larch.

On the basis of a large amount of work, 41 candidate larch promoters are searched after the second generation transcriptome sequencing and the third generation genome sequencing of the larch are carried out. Then, 6 candidate larch endogenous promoters predicted to be excavated are found in the 41 candidate promoters, the numbers of the candidate promoters are LarPE004, LarPE005, LarPE051, LarPE055, LarPE057 and LarPE058 respectively, and the ZmUbi1 promoter from corn can successfully start the transient expression of green fluorescent protein in larch cells, and has certain transcription functional activity. It was also unexpectedly found that the activity of LarPE004 was significantly higher than all other promoters in the same experiment. Then, the transcription activity of the LarPE004 and CaMV35S promoters is studied in rice to verify the universality and the transcription activity of the LarPE004 promoter. The result shows that the promoter LarPE004 still has stronger functional activity after being integrated into the genome in rice.

Therefore, the present invention provides a novel Larix Gmelini-derived promoter (the nucleotide sequence of which is shown in Seq ID No. 1). Can not only drive gene expression in larch efficiently and stably, but also drive gene expression in other plant species such as rice efficiently and stably.

The present invention will be further described with reference to the following specific examples.

Example 1 search and analysis of Larix Gmelini candidate promoters

6g of callus of Larix kaempferi (Larix kaempferi) cultured for 14 days after induction culture was equally divided into 3 parts, and 6g of coniferous needle of Larix kaempferi cultured for 42 days was equally divided into 3 parts, which were used for transcriptome second-generation sequencing and genome third-generation sequencing, respectively. Flux sequencing was performed by bekkiso biotechnology limited. By utilizing a bioinformatics technology, firstly, splicing and assembling the third-generation sequencing result of the larch genome to obtain genome sequence information, then carrying out transcriptomics analysis according to the RNA-seq result, and calculating to obtain the expression quantity information of the gene in the larch callus and the needle leaf.

Since promoters are important functional elements for controlling gene transcription expression, plant promoters should theoretically be rich in numerous transcription regulatory elements and also rich in conserved sequences of core elements, such as TATA box, CAAT box, etc. Using plant Care (M.Lescot, P.D. hais, G.Thijs, et al.plant CARE, a database of plant cis-acting regulatory elements and a port to tools for in silico analysis of promoter sequences [ J ]. Nucleic Acids Research,2002,30(1):325-327), the studies predicted the cis-acting and core element distribution of all candidate larch endogenous promoters.

The plantarcae analysis results showed that the 41 candidate larch endogenous promoters contained numerous cis-acting elements involved in transcriptional regulation. The cis-acting elements of transcriptional regulation distributed on the larch promoter are related to auxin response elements, light response elements, drought stress response elements, low temperature stress response elements, abscisic acid response elements, wound response elements and the like, and the determined excavation range is really a promoter region.

Example 2 isolation and identification of Larix Gmelini candidate promoter and construction of expression vector

To verify the functional activity of these 41 predicted candidate larch endogenous promoters, the fluorescent reporter vector pGEL055(Ren Q, Zhong Z, Wang Y, et al. bidirectional Promoter-Based CRISPR-Cas9 Systems for Plant Genome Editing. front Plant Sci.2019; 10:1173.doi:10.3389/fpls.2019.01173) was selected as the backbone vector. In order to facilitate the assembly of the 41 candidate larch endogenous promoters waiting for activity verification and the incorporation of the most commonly used constitutive promoter CaMV35S promoter in plants into the backbone vector, the present study added AvrII restriction endonuclease sites at the 5 'end and PstI restriction endonuclease sites at the 3' end of the ZmUbi1 promoter of the backbone vector pGEL055, and fused the β -glucuronidase Gene (GUS) with the green fluorescent protein Gene (GFP) to obtain the expression vector pLB 41. The amplified promoter fragment and the fragment obtained after the enzyme digestion of the skeleton vector can be bridged by virtue of homologous arms to realize batch Gibson assembly, and the cloning of 41 promoters to be verified and the CaMV35S promoter is respectively completed.

The specific operation of the promoter separation is to use larch genome DNA as a template, design specific primers according to DNA sequences at two ends of a 2000bp region of a candidate promoter, and respectively amplify early-stage excavation from larch genome DNA through touchdown PCR to obtain 41 candidate larch endogenous promoter sequences with potential research values.

Wherein the amplification primers of LarPE004 are as follows:

5-end primer: lkpro004-F (SEQ ID No. 2):

ctgaattaacgccgaattaattcctaggTTCCTTTTTTGGACTATTTTATCATAAAAG；

3-end primer: lkpro004-R (SEQ ID No. 3):

ACTGGGCCATtTTTTTtctagaCTGCAGATATTGAAGTTTTCAATAAAGAATAACCCA。

after the amplified promoter region DNA fragments were recovered by cutting gel and ligated to the backbone vector, 33 of them were successfully cloned and constructed as fluorescent reporter vectors that could be used to detect promoter activity. Enzyme digestion and sequencing verification prove that vectors with 35 promoters (including ZmUbi1(pLB41) and CaMV35S promoter (pLB42)) are successfully obtained, and a schematic diagram of an expression vector is shown in figure 1.

Example 3 comparative analysis of transcriptional Activity of Larix Gmelini candidate promoters

Compared with the traditional transformation, the transient transformation of the protoplast greatly shortens the detection period, reduces the operation difficulty and improves the experimental efficiency. Therefore, the instant expression system of the larch protoplast is applied in the experiment to detect the transcription functional activity of 33 larch endogenous candidate promoters successfully constructed in the vector in example 2. Transient transformation of larch protoplasts was performed sequentially on the 35 fluorescent reporter expression vectors obtained in example 2, and after 48h of dark culture, transient expression of green fluorescent protein was observed under an inverted fluorescent microscope to determine transcriptional activity of candidate promoters. The results of the detection are shown in FIG. 2. Through observation, 6 candidate larch endogenous promoters predicted to be excavated are found, the numbers of the candidate larch endogenous promoters are LarPE004, LarPE005, LarPE051, LarPE055, LarPE057 and LarPE058, and the ZmUbi1 promoter (pLB41) from corn successfully starts the transient expression of green fluorescent protein in the larch protoplast cells, so that the larch endogenous promoters have certain transcription functional activity. Meanwhile, the transcriptional activity of the LarPE004 promoter is found to be obviously superior to that of the ZmUbi1(pLB41) and CaMV35S promoter (pLB42) which are commonly used in the current plant genome engineering in all expression results.

Example 4 transient expression of Rice protoplasts to verify LarPE004 transcriptional Activity

Furthermore, the invention carries out protoplast transient transformation in rice to verify the universality and the transcription activity of the LarPE004 promoter. The specific operation is to transfer the pLB _ LarPE004 expression vector and pLB41 and pLB42 two control vectors into rice protoplast cells for transient expression. After 48h of dark culture, the GFP expression level was measured. Microscopic observation of a fluorescence microscope shows that the LarPE004 promoter still keeps high transcription activity in rice, and as shown in figure 3, the LarPE004 promoter is proved to have genome engineering application potential.

Example 5 transformation of Agrobacterium Rice to verify LarPE004 transcriptional Activity

Then, transformation experiments are carried out in rice to verify the universality and the transcription activity of the LarPE004 promoter. The specific operation is that the two expression vectors of pLB _ LarPE004 and pLB42 are respectively introduced into the rice genome by an agrobacterium transformation method to carry out a rice stable transformation experiment. Firstly, observing the expression condition of green fluorescent protein of the rice neogenetic callus transformed by the agrobacterium under a stereoscopic fluorescent microscope. It was found that the calli of rice transformed with the pLB _ LarPE004 and pLB42 (loaded with CaMV35S promoter) vectors all emitted green fluorescence, as shown in FIG. 4. This initially demonstrated that the excavated larch endogenous LarPE004 promoter remains functionally active after integration into the plant genome.

In order to further verify the functional activity of the LarPE004 promoter, after the stably transformed rice plant grows and roots, rice leaves and root tissues transferred into the pLB _ LarPE004 vector are taken out, and a GUS histochemical staining experiment is carried out. After 24h, the leaves and the root tissues of the rice plant are found to have no color change compared with wild rice plants, the root tissues and the leaves of the rice plant which is transferred into the pLB _ LarPE004 vector are dyed blue, as shown in figure 5 (only the result after the root tissues of the rice plant are dyed is shown in the figure), and the LarPE004 promoter is proved to be capable of promoting the stable expression of the GUS gene in the transformed rice plant.

Sequence listing

<120> larch endogenous efficient promoter and application

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1953

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttcctttttt ggactatttt atcataaaag ttaatttcat tctagtcttc ccctcttctt 60

ttatacaatt ttattattaa ttgtctaaca actaaattct ttaacaacca atatatttca 120

tccatcgact ttctcttatc tatcttacaa tcttatatgg gtcacgtaat gatggatgga 180

gtgtccactt gaaacattgg atcatttcaa aacaaataaa aaataaaatt atagaaaatt 240

ttattgcaaa aaaattacaa aaatgtacta gtaaaaaaga ttaaatttat ccatttatta 300

actcactcca ttcctcccta cctacttatg ggaacctatt cattttacct ccttatggga 360

ccctctattc attttacaca ccattattca ttaggaagat tggatgtatg cacattgaat 420

cccaaccatg gtgggcaagg gaggagggca ggtgtttata aagaaaaaga gaaaatttat 480

taatatcgtg gttatcttga agacgaacct aaggaaaaac tcaaaaacat taaaataatg 540

aaaggtacta ccacaacttt tcccattctt gatgccaatg caagttagtt cctttcttat 600

acttggcatg ccaatactta cttgagaaaa aagtaataaa gtgatagaat ggtcaataat 660

tttgaacact aaggctccca cataatttgt ttatacttat cattaaaaaa aataagaaca 720

taacccctaa ggcacataat ttgttttccc tcatcattta aaaaaaagag gaacataact 780

cctaaagaat accttatcac aacttgatag agatatcaat tgatgataaa gaaggtctaa 840

ccctatttat atctccccct caagtccttg ccttgtgggc taagtaaaag gctttggtgc 900

aaggagcttc tagagggggc ggtgacttat ttatttatta ttataattat ttttaaaata 960

aggaccggcc ctattattta tttattatta taattatttt taaaataatt tatttaatta 1020

ttaactattt gtttgtgtgg ttactttcaa cacaatgtat ctagacactt tcggtcaatt 1080

attgaccaaa atttaagctt taagataatt atttgatgca attgatgtgg taaatgatca 1140

tcagtttggc atgtgcccaa ccactacaga gcccgcggga ccaagcaacc ttatcttttc 1200

ttcgttacgg agccaatcaa aattttagaa gggagaatac tttaaggcgg ctccaccgac 1260

cttatcccac ttgcacaagt ggcctcctag aagctgacac ctgtcttaat atgaatggac 1320

ttcattgggc gggtcgtcga tatggtgaat ttaaatacgg ctccacccct cattattcat 1380

tgtgatttct tgatttggag agttttccac gaacggcagg cgaagcaacc gagagcgtct 1440

ctatcgattg agttcaggta acgtgttctt cgtctcgatt tctttctttg tattttttaa 1500

tgtctctggt tctcatttaa ctgtggtagg cttgctcttc ttgatcttcg ttttgaatcc 1560

caaatcagaa ggctattctc cggatctcgt tttgaattcc aaatcagagg tttttatttc 1620

gttattgtga tttattttct ggaattattg ttttgataaa aggttttcgt ttaatttcat 1680

cggttatggc ttgatgagga ataccaaatc ctatttgctt cttgtgtgat ttgttgttcc 1740

tctatttctg ggttttaatg gagttatttg ccagtttgtg ttttataggg ttttgaatcc 1800

gtgaatcctc gacagcttgt agcctagggt tttattttgc atctgcgggt tattatcatt 1860

tattgttcag gttttcagac tatccttatt gtttctgact atttcttatg gttgtgcagg 1920

tggtgggtta ttctttattg aaaacttcaa tat 1953

<210> 2

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctgaattaac gccgaattaa ttcctaggtt ccttttttgg actattttat cataaaag 58

<210> 3

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

actgggccat tttttttcta gactgcagat attgaagttt tcaataaaga ataaccca 58

Claims

A DNA molecule characterized in that said DNA molecule is a) or b) or c) as follows:

a) DNA molecule shown in SEQ ID No. 1;

b) a DNA molecule having a homology of 99% or more, 95% or more, or 90% or more with the nucleotide sequence defined in a) and having a promoter function;

c) a DNA molecule which is hybridized with the nucleotide sequence defined by a) or b) under strict conditions and has the function of a promoter.
2. A gene expression cassette comprising a DNA molecule according to claim 1 or a recombinant plasmid comprising a DNA molecule according to claim 1.
3. A recombinant microorganism, transgenic plant cell line or transgenic animal cell line comprising the DNA molecule of claim 1.
4. Use of the DNA molecule of claim 1 as a promoter.
5. Use of a DNA molecule according to claim 1, an expression cassette according to claim 2 or a recombinant plasmid according to claim 3 for promoting expression of a gene of interest.
6. Use according to claim 6, characterized in that the promotion of expression of a gene of interest is the promotion of expression in a microorganism, a plant cell or an animal cell.
7. Use according to claim 6, characterized in that said plant is a gymnosperm or an angiosperm.
8. Use according to claim 6, characterized in that gymnosperms are plants of the Pinaceae family; preferably, the Pinaceae plant is a Larix plant;

alternatively, the angiosperm is a monocot; preferably, the monocotyledon is a gramineae plant.
9. A method for expressing a target gene, which comprises using the DNA molecule of claim 1 as a promoter to promote the expression of the target gene.
10. The method of claim 9, wherein the specific DNA molecule of claim 1 is operably linked upstream of the gene of interest to be expressed, thereby promoting expression of the gene of interest.