CN113943733B

CN113943733B - Larix Gmelini endogenous promoter and application

Info

Publication number: CN113943733B
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: 2023-06-09
Anticipated expiration: 2040-06-30
Also published as: CN113943733A

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a DNA molecule with a promoter function of larch endogenous and application thereof. The technical problem of the invention is to provide a larch endogenous high-activity starting element. The technical scheme for solving the technical problem is to provide a DNA molecule. The DNA molecule is represented by the following a) or b) or c): a) 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 can be complementarily paired with the nucleotide sequence defined in a) or b) and has the function of a promoter. Experiments prove that the larch endogenous promoter can not only efficiently and stably drive gene expression in larch, but also efficiently and stably drive gene expression in other plant species such as rice and the like, and has good application prospect.

Description

Larix Gmelini endogenous promoter and application

Technical Field

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

Background

Larch is a type of larch, belonging to the family Pinaceae (Pinaceae), genus larch (Larix). Larch is widely distributed in mountain areas, plain areas of temperate zones and mountainous climates, and 25 larches are all distributed in the global scope. The species number of larch genus in our world is known as fescue, wherein larch (larx principles-rupprechtii), larch (larx gmelinii), larch (larx kaempferi) and larch (larx olgensis) are mainly distributed in northeast and northwest regions of our country; siberian larch (Larix sibirica) is mainly distributed in the North-west Xinjiang Altaishan region of China. Larch has the characteristics of quick growth at early stage, 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 construction of protective forests and to cultivation and returning projects. Because the growth of the forest is a process with extremely long period, the research of early people around larch is mainly focused on the traditional biological aspects of genetic variation research, seed source selection, woodiness property research, photosynthetic characteristic research and the like, and greatly restricts the genetic property improvement process of the forest.

The advent of genomic engineering techniques such as transgenic technology and genome editing has brought new opportunities and challenges for improvement of larch genetic traits. The transgenic technology is a technology of introducing exogenous DNA with complete expression unit designed in advance into receptor cells by biotechnology means and integrating the exogenous DNA on receptor genome, so that the exogenous gene can be stably expressed in the receptor and the transgenic technology also has the capability of generation inheritance. The genome editing technology is to modify nuclease, introduce into acceptor cell and combine or cut at the selected position of genome to form double strand break, so as to realize site-directed gene knockout or expression regulation of downstream gene. 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 trait of larch, thereby obtaining She Songshu plants which grow rapidly, have high yield and high quality stress resistance.

Promoters are important functional elements for regulating the transcriptional expression of downstream genes, and are a piece of DNA located upstream of the start codon (ATG) of the gene, capable of binding to RNA polymerase and initiating the transcription of the gene, corresponding to the "switch" of gene transcription. The promoter is a key factor for driving target genes to be expressed and translated in cells, and the gene engineering technology is realized by using the promoter, so that the larch endogenous promoter with excellent digging activity is a problem to be solved in the research of directional genetic improvement and other aspects of larch by utilizing the modern biological technologies such as a transgenic technology or a genome editing technology. However, since the current larch reference genome sequence map assembly is not completed, the genome analysis work of the larch is less in progress, the information of functional elements of an endogenous promoter is less, and the digging of the promoter with excellent endogenous activity of the larch is difficult.

Disclosure of Invention

The invention aims to provide a larch endogenous high-activity starting element. The technical scheme for solving the technical problem is to provide a DNA molecule. The DNA molecule is represented by the following a) or b) or c):

a) 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 can be complementarily paired with the nucleotide sequence defined in 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 recombinant microorganisms, transgenic plant cell lines or transgenic animal cell lines containing the above DNA molecules.

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

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

Wherein the initiation of the expression of the target gene is initiated in a microorganism, a plant cell or an animal cell.

Wherein the plant described in the above application is gymnosperm or angiosperm.

Further, the gymnosperm is a pinaceae plant. Preferably, the pinaceae plant is a larch genus plant. Still further, the larch is at least one selected from larch (larx principles-rupprechiii), larch (larx gmelini), larch (larx kaempferi) and larch (larx olgensis).

Wherein the angiosperm described in the above application is a monocotyledonous plant. Further, the monocotyledonous plant is a plant of the Gramineae family. Wherein the gramineous plant is a oryza plant. Further, the rice plant is rice.

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

Wherein, in the above method, the above DNA molecule is operably linked upstream of the target gene to be expressed, and the expression of the target gene is initiated.

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

Drawings

FIG. 1, schematic diagrams of framework vectors and expression vectors used in larch promoter mining and expression detection experiments.

FIG. 2, transient transformation of larch protoplasts with green fluorescent protein expression (objective 10X).

FIG. 3, green fluorescent protein expression after transient transformation of rice protoplasts (objective lens 10X).

Fig. 4, rice callus green fluorescent protein expression (scale bar=1 mm).

FIG. 5, results of GUS histochemical staining of transgenic rice roots (scale bar=1 cm).

As the promoter is used as an important functional element for regulating gene transcription expression, the identification of the promoter with excellent endogenous activity of larch creates great value for the work of cultivating new larch varieties, transgenic research and the like. However, the high-quality reference genome sequence of larch is not finished at present, and the excavation work of the endogenous promoter of larch is difficult.

On the basis of a large amount of work, 41 candidate larch She Songqi movers are found after the second generation transcriptome sequencing and the third generation genome sequencing of larch are performed. Then, 6 candidate larch endogenous promoters which are predicted and mined are found in 41 candidate promoters, and the numbers of the candidate larch endogenous promoters are LarPE004, larPE005, larPE051, larPE055, larPE057 and LarPE058 respectively, and the candidate larch endogenous promoters and the ZmUbi1 promoter from corn can successfully start the instant expression of green fluorescent protein in larch cells, and the candidate larch endogenous promoters have a certain transcription function activity. It was also unexpectedly found that the activity of LarPE004 was significantly higher than all other promoters in the same experiment. And then, carrying out transcriptional activity research on LarPE004 and CaMV35S promoters in rice to verify the universality and transcriptional activity of the LarPE004 promoters. As a result, it was found that the promoter LarPE004 still has a strong functional activity after integration into the genome in rice.

Thus, the present invention provides a novel larch-derived promoter (the nucleotide sequence of which is shown in Seq ID No. 1). 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.

The invention is further illustrated by the following examples.

Example 1 search and analysis of larch candidate promoters

6g of larch (Larix kaempferi) callus cultured for 14 days under induction was aliquoted into 3 parts, and 6g of larch needle cultured for 42 days was aliquoted into 3 parts for respectively performing the second generation sequencing of transcriptome and the third generation sequencing of genome. Throughput sequencing was performed by Baimeike Biotechnology Inc. By utilizing bioinformatics technology, firstly, splicing and assembling results obtained by three-generation sequencing of larch genome to obtain genome sequence information, then, carrying out transcriptome analysis according to an RNA-seq result, and calculating to obtain expression quantity information of genes in larch callus and needle leaves.

Since promoters are an important class of functional elements for controlling transcriptional expression of genes, plant promoters should be theoretically rich in many transcriptional regulatory elements, and should also be rich in conserved sequences of core elements, such as TATA boxes, CAAT boxes, etc. Using the online promoter analysis software of Plantare (M.Lescot, P.Dhais, G.Thijs, et al plant CARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences [ J ]. Nucleic Acids Research,2002,30 (1): 325-327), studies were made to predict the distribution of cis-acting elements and core elements of all candidate endogenous promoters of Larix Gmelini.

The plantacare analysis showed that these 41 candidate endogenous larch promoters contained a number of cis-acting elements involved in transcriptional regulation. Transcription regulatory cis-acting elements distributed on the selected She Songqi promoter relate 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 indicate that the excavation range determined by the invention is really a promoter region.

Example 2 isolation and identification of larch candidate promoters and construction of expression vectors

To verify the functional activity of these 41 predicted candidate endogenous promoters of larch, the fluorescence reporting vector pGEL055 (Ren Q, zhong Z, wang Y, et al, bidirectory Promoter-Based CRISPR-Cas9 Systems for Plant Genome edition.front Plant Sci.2019; 10:1173.doi:10.3389/fpls.2019.01173) was selected as the backbone vector. To facilitate the assembly of these 41 candidate endogenous promoters to be validated separately, and the constitutive promoter CaMV35S promoter most commonly used in plants into the backbone vector, the study added an AvrII restriction enzyme site at the 5 'end and a PstI restriction enzyme site at the 3' end of the ZmUbi1 promoter of the backbone vector pGEL055, and fused the beta-glucosidase Gene (GUS) with the green fluorescent protein Gene (GFP), yielding expression vector pLB41. The amplified promoter fragments and the fragments after the backbone vector enzyme cutting can be bridged by means of homologous arms, so that batch Gibson assembly is realized, and the cloning of 41 promoters to be verified and CaMV35S promoters is respectively completed.

The specific operation of promoter separation is to design specific primers according to DNA sequences at two ends of a 2000bp region of a candidate promoter by taking larch genomic DNA as a template, and respectively amplifying 41 candidate larch endogenous promoter sequences with potential research value from the larch genomic DNA by landing PCR.

The amplification primers of LarPE004 are as follows:

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

ctgaattaacgccgaattaattcctaggTTCCTTTTTTGGACTATTTTATCATAAAAG；

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

ACTGGGCCATtTTTTTtctagaCTGCAGATATTGAAGTTTTCAATAAAGAATAACCCA。

the amplified DNA fragment of the promoter region is recovered by cutting gel, and after being connected with a skeleton carrier, 33 successfully cloned and constructed into a fluorescence report carrier which can be used for detecting the activity of the promoter. The enzyme digestion and sequencing verification prove that a vector containing 35 promoters (including ZmUbi1 (pLB) and CaMV35S promoter (pLB)) is successfully obtained, and the schematic diagram of the expression vector is shown in figure 1.

Example 3 comparative analysis of transcription Activity of larch 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, this experiment uses a larch protoplast transient expression system to examine the transcriptional functional activity of 33 endogenous candidate promoters of larch successfully constructed in the vector in example 2. The 35 fluorescence report expression vectors obtained in the above example 2 were sequentially subjected to transient transformation of larch protoplasts, and after 48 hours of dark culture, the transient expression of green fluorescent protein was observed under an inverted fluorescent microscope for microscopic examination to determine the transcription activity of the candidate promoters. The detection results are shown in FIG. 2. Through observation, 6 candidate larch endogenous promoters which are predicted and excavated are found, the numbers of the candidate larch endogenous promoters are LarPE004, larPE005, larPE051, larPE055, larPE057 and LarPE058, and the candidate larch endogenous promoters from corn (pLB 41) successfully start the instant expression of green fluorescent protein in the larch protoplast cells, and the candidate larch endogenous promoters have a certain transcription function activity. Meanwhile, in all the expression results, the transcriptional activity of the LarPE004 promoter is obviously superior to that of ZmUbi1 (pLB 41) and CaMV35S promoter (pLB) which are commonly used in the current plant genome engineering.

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

Furthermore, the invention carries out transient transformation of protoplast in rice to verify the universality and transcriptional activity of LarPE004 promoter. Specifically, pLB _LarPE004 expression vectors and pLB, pLB and two control vectors are transferred into rice protoplast cells for transient expression. After 48h of dark culture, GFP expression levels were detected. The fluorescence microscopy observation shows that the LarPE004 promoter still maintains high transcriptional activity in rice, and as shown in figure 3, the LarPE004 promoter has genome engineering application potential.

Example 5 transformation by Agrobacterium tumefaciens to verify LarPE004 transcriptional Activity

Then, transformation experiments are carried out in rice to verify the universality and transcriptional activity of the LarPE004 promoter. Specifically, the pLB _LarPE004 and pLB42 expression vectors are respectively introduced into rice genome by an agrobacterium transformation method to perform rice stable transformation experiments. Firstly, observing the expression condition of green fluorescent protein of the rice new-born callus after agrobacterium transformation under a stereoscopic fluorescent microscope. It was found that both the pLB _LarPE004 and pLB (CaMV 35S promoter loaded) vector transferred rice calli emitted green fluorescence, as shown in FIG. 4. This preliminary demonstrates 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 plants grow into seedlings and root, the rice leaves and root tissues transferred into the pLB _LarPE004 vector are taken out, and GUS histochemical staining experiments are carried out. After 24 hours, compared with the wild rice plant leaves and root tissues which have no color change, the rice root tissues and leaves transferred into the pLB _LarPE004 vector are dyed blue, as shown in figure 5 (only the result after the rice root tissues are dyed is shown in the figure), and the LarPE004 promoter can start GUS gene stable expression in the transformed rice plant.

Sequence listing

<120> Larix Gmelini endogenous Gao Xiaoqi 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 the nucleotide sequence of said DNA molecule is shown in SEQ ID No. 1.
2. A gene expression cassette comprising the DNA molecule of claim 1 or a recombinant plasmid comprising the DNA molecule of claim 1.
3. A recombinant microorganism comprising the DNA molecule of claim 1.
4. Use of the DNA molecule of claim 1 as a promoter.
5. Use of the DNA molecule of claim 1, the expression cassette of claim 2 or the recombinant plasmid of claim 2 to initiate expression of a gene of interest.
6. The use according to claim 5, wherein said initiation of the expression of the gene of interest is an initiation of expression in a microorganism or a plant cell.
7. The use according to claim 6, characterized in that the plant is a gymnosperm or an angiosperm.
8. Use according to claim 7, characterized in that the gymnosperm is a pinaceae plant.
9. Use according to claim 8, characterized in that the pinaceae plant is a larch genus plant.
10. The use according to claim 7, characterized in that the angiosperm is a monocotyledonous plant.
11. Use according to claim 10, characterized in that said monocotyledonous plant is a plant of the Gramineae family.
12. A method for expressing a target gene, comprising using the DNA molecule of claim 1 as a promoter to promote expression of the target gene.
13. The method according to claim 12, characterized in that the DNA molecule according to claim 1 is operably linked upstream of the gene of interest to be expressed, initiating the expression of the gene of interest.