CN114717210A

CN114717210A - Poplar geranylgeraniol reductase and coding gene and application thereof

Info

Publication number: CN114717210A
Application number: CN202210365792.7A
Authority: CN
Inventors: 何方; 陈良华; 时羽杰; 张帆
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-04-08
Filing date: 2022-04-08
Publication date: 2022-07-08
Anticipated expiration: 2042-04-08
Also published as: CN114717210B

Abstract

The invention provides a poplar geranylgeraniol reductase, a coding gene and application thereof, wherein the geranylgeraniol reductase is derived from poplar, and the amino acid sequence of the geranylgeraniol reductase is shown as SEQ ID NO. 12. The reductase gene PtrCHLP3 is used for constructing transgenic poplar, and the growth rate and leaf color characteristics of poplar are initially changed in a directional mode. The PtrCHLP3 gene can slow down the growth of plant, reduce the plant height, thin the stem, yellow the leaf, etc. and provides new method for initial breeding of new poplar variety.

Description

Poplar geranylgeraniol reductase and coding gene and application thereof

Technical Field

The invention relates to the technical field of plant genes, in particular to a poplar geranylgeraniol reductase, and a coding gene and application thereof.

Background

Chlorophyll molecules are ubiquitous in photosynthetic organisms, which complete the basic process of capturing light energy in an antenna system by driving electron transfer in the reaction center. Chlorophyll plays an important role in photosynthetic organisms such as plants. Chlorophyll a is the main electron donor for PSI and PSII reaction centers. With the development of molecular biology and protein structure, key members involved in the chlorophyll biosynthesis pathway have been fully explored and identified. Geranylgeraniol reductase (CHLP) plays an important role in plant photosynthesis and chlorophyll synthesis and is involved in the terminal hydrogenation step of chlorophyll biosynthesis. Currently, the CHLP gene has been identified in many species, including photosynthetic bacteria, algae, tobacco, rice, peach, olive, and tomato. The chlp mutant of cyanobacteria has reduced chlorophyll and carotenoid content. After the CHLP gene of the tobacco is silenced, the plant grows slowly, and leaves are pale or mottled. The leaves of the chlp mutant in rice are yellow-green, which affects plant growth. In poplar, the PtrCHLP3 gene plays an important role in regulating the growth rate, photosynthetic rate, leaf color and other aspects of poplar. Therefore, the screening and cloning of the PtrCHLP3 gene and the genetic transformation by using the PtrCHLP3 gene are one of the important ways to improve new plant varieties and create colored-leaf plants.

The colored-leaf tree species are important material bases for building park cities. However, the current color-leaf garden trees are single and scarce in variety and can not meet the requirements of urban landscaping, so that the cultivation of new color-leaf tree varieties becomes an important target of garden tree breeding. Therefore, a rapid and directionally improved molecular breeding technology is urgently needed to make up for the defects of the traditional garden tree breeding technology and efficiently select and breed new varieties of colored-leaf trees so as to meet the requirements of park city construction. The existing molecular breeding aiming at color transformation mainly focuses on the transformation of the flower color of herbaceous flowers, and the molecular breeding of color-leaf trees is almost blank at home and abroad. Because the key genes forming the leaf color of the colored-leaf trees and the regulation mechanism thereof are not known clearly, the defects and the bottleneck greatly obstruct the molecular breeding process of the colored-leaf trees, and thus there is a report that the leaf color is reformed by a molecular biological means to breed a new variety of the colored-leaf trees. At present, the research on the PtrCHLP3 gene of the poplar is not reported, and the technology for improving new varieties of poplar and creating colored-leaf plants by using the gene is lacked. If the expression of the PtrCHLP3 gene can be regulated and controlled by the genetic engineering technology, the growth rate and the leaf color of the poplar can be gradually improved, the application of the poplar in production can be greatly promoted, and the method has very important practical significance for forest molecular breeding research and forestry ecological civilization construction.

Disclosure of Invention

Aiming at the problems and defects in the prior art, the invention provides a poplar geranylgeraniol reductase as well as a coding gene and application thereof. The technical scheme of the invention is as follows:

in a first aspect, the invention provides geranylgeraniol reductase derived from poplar, wherein the amino acid sequence of geranylgeraniol reductase is shown as SEQ ID No. 12.

In a second aspect, the invention also provides a biomaterial related to the geranylgeraniol reductase, which is any one of the following 1) to 5):

1) a nucleic acid molecule encoding geranylgeraniol reductase;

2) an expression cassette comprising 1) the nucleic acid molecule;

3) a recombinant vector comprising 1) said nucleic acid molecule;

4) a recombinant microorganism containing 1) said nucleic acid molecule;

5) a recombinant microorganism comprising 3) the recombinant vector.

Further, the nucleotide sequence of the nucleic acid molecule for coding geranylgeraniol reductase is shown as SEQ ID NO. 1.

In a third aspect, the invention provides an application of the geranylgeraniol reductase or the biological material in regulation of growth and development of poplar.

Further, the regulation of the growth and development of the poplar comprises regulation of the growth rate and/or the leaf color of the poplar.

Further, the regulation comprises inhibiting the expression of the geranylgeraniol reductase or the biological material in the poplar so as to slow the growth rate of the poplar and/or yellow the leaf color.

In a fourth aspect, the invention provides a method for constructing a transgenic poplar, comprising the following steps:

(1) taking a gene sequence of a poplar geranylgeraniol reductase coding gene PtrCHLP3 shown in SEQ ID NO.1 as a template, and respectively amplifying a forward fragment and a reverse fragment by using a forward fragment cloning primer and a reverse fragment cloning primer;

(2) taking PCAMBIA2301-PS as a skeleton vector, carrying out double enzyme digestion (SacI/XbaI) on the pCAMBIA2301-PS vector, and then sequentially recombining a forward sequence, an RTM sequence and a reverse sequence of a partial fragment of PtrCHLP3 into the PCAMBIA2301-PS vector to obtain a pCAMBIA2301-PtrCHLP3-RNAi positive plasmid;

(3) the pCAMBIA2301-PtrCHLP3-RNAi positive plasmid is used for transforming poplar by a leaf disc method.

Further, the forward fragment cloning primers in the step (1) are PtrCHLP3-F1 and PtrCHLP3-R1, and the reverse fragment cloning primers are PtrCHLP3-F2 and PtrCHLP 3-R2; the sequence of the forward fragment cloning primer PtrCHLP3-F1 is shown as SEQ ID No.2, the sequence of the forward fragment cloning primer PtrCHLP3-R1 is shown as SEQ ID No.3, the sequence of the reverse fragment cloning primer PtrCHLP3-F2 is shown as SEQ ID No.4, and the sequence of the reverse fragment cloning primer PtrCHLP3-R1 is shown as SEQ ID No. 5.

Further, in the step (3), the transformation of poplar with pCAMBIA2301-PtrCHLP3-RNAi expression vector by leaf disc method specifically comprises: transforming the pCAMBIA2301-PtrCHLP3-RNAi positive plasmid into expression strain competence, infecting poplar leaf disc by an infection method, and screening positive transgenic strains to obtain transgenic poplar.

Preferably, the expression strain is agrobacterium.

The invention has the beneficial effects that:

the invention provides a new application of a PtrCHLP3 gene of a poplar in regulating the growth and development of the poplar, in particular regulating the growth speed, the photosynthetic rate and the leaf color of the poplar. And further adopting a transgenic technology to integrate a vector containing a partial fragment of the PtrCHLP3 gene into a poplar genome, and primarily directionally changing the growth rate and leaf color characteristics of the poplar. The PtrCHLP3 gene can slow down the growth of plant, reduce the plant height, thin the stem, yellow the leaf, etc. and provides new method for initial breeding of new poplar variety.

Drawings

FIG. 1 is a diagram showing the electrophoresis chart of the PtrCHLP3 expression vector constructed in example 1; (a) carrying out total RNA electrophoresis detection; (b) full-length electrophoretogram of target fragment; 1-4: full-length fragment of target gene PtrCHLP 3; m is Mark 2000; (c) PCR verifies the agrobacterium transformation electrophoretogram M, Mark 2000; 1-6: PCR verification of pCAMBIA2301-PtrCHLP3-RNAi recombinant plasmid;

FIG. 2 is the vector map of expression-suppressing plant expression vector pCAMBIA2301-PtrCHLP3-RNAi constructed in example 1 of the present invention.

FIG. 3 is a diagram showing transformation, regeneration and phenotype of a PtrCHLP3 transgenic poplar in example 1 of the present invention; wherein, a: callus tissue; b: regenerating a transgenic bud cluster by using a leaf disc; c: a regenerated resistant transgenic plantlet; d: a phenotype graph of a control (WT) and an expression-inhibition (L1-L9) transgenic tender plant for subculture cuttage for 30 days;

FIG. 4 is a diagram showing different modes of detection of a PtrCHLP3 transgenic poplar in example 1 of the present invention; (a) the constructed expression-inhibiting plant expression vector pCAMBIA2301-PtrCHLP3-RNAi schematic diagram fragment; (b) detecting an electrophoretogram by using a specific primer of the transgenic poplar with the PtrCHLP3 gene; m: mark 2000; PC: p2301-PnCHLP-RNAi positive control vector; NC: negative control; WT: a wild type; L1-L9: PtrCHLP3 inhibits the expression of poplar transgenic lines; (c) histochemical GUS staining patterns of different transgenic plants; WT: a wild type; L1-L9: PtrCHLP3 inhibits the expression of poplar transgenic lines; (d) analyzing the relative expression quantity of the PtrCHLP3 genes of different transgenic lines; WT: a wild type; L1-L9: PtrCHLP3 inhibits the expression of poplar transgenic lines;

FIG. 5 is a phenotypic graph of transgenic poplar with PtrCHLP3 gene and chlorophyll content, as shown in example 1; (a) a top view of poplar leaves; WT: a wild type; chlp-3/4: PtrCHLP3 inhibits the expression of poplar transgenic lines; (b) chlorophyll content of poplar leaves; and (3) Chla: chlorophyll a; and (3) Chlb: chlorophyll b; and (6) Caro: a carotenoid;

FIG. 6 is a 35-day statistical analysis of the phenotype of seedlings from a control (WT), expression-repressed (chlp-3/4) positive line in example 1 of the present invention. (a) The method comprises the following steps A performance map; (b) the method comprises the following steps The relationship between plant height and days; (c) the method comprises the following steps Fresh weight of different parts; (d) the method comprises the following steps The stem growth amount; (e) the method comprises the following steps Ground diameter; (f) - (k): 8, cross section staining pattern of the stem of the third node; (i) - (m): and (8) carrying out statistics on the cross section data of the stem of the 8 th node.

Detailed Description

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1

1. Obtaining PtrCHLP3 Gene

The total RNA extraction material is young leaves of 84K poplar, liquid nitrogen is adopted for full grinding, the method is carried out according to the specification of an RNA extraction Kit of RNAprep Pure Plant Kit of Alelix biological company in Hunan, RA-10 enucleation enzyme reagent is used in a matched mode in the experiment process for spraying an experiment instrument and an operation table, and RNase remained on the surface is removed. The concentration of RNA, OD260/OD280, and OD260/OD230 were measured using a Thermo Nanodrop 2000 nucleic acid/protein quantitation instrument, and the absorption peak of the sample at 260nm was observed. RNA integrity was checked by electrophoresis under the conditions of 150V, 20min and 1% agarose gel, and the result is shown in a in FIG. 1. Synthesis of cDNA reference is made to Alelix organisms of Hunan province

All-in-One First-Strand cDNA Synthesis Supermix for PCR kit instructions. Obtaining the nucleotide sequence of CHLP3 gene of poplar according to populus tomentosa genome in populus tomentosa database, and designing Primer PtrCHLP3-F1 (the sequence is shown as SEQ ID NO) by using Primer 5.02) and PtrCHLP3-R1 (the sequence is shown as SEQ ID NO. 3), the cDNA synthesized in the previous step is taken as a template, and the full length of the gene is amplified by PCR. 25 μ L reaction:

supermix 12.5. mu.L, primer F (10. mu.M) 1. mu.L, primer R (10. mu.M) 1. mu.L, cDNA template 2. mu.L, ddH₂O8.5 mu L; reaction procedure: pre-denaturation at 94 ℃ for 3min, 35cycles (94 ℃ for 30s, 62 ℃ for 30s, 72 ℃ for 1min), extension at 72 ℃ for 5 min; storing at 4 ℃. After the PCR reaction, the product was detected by 1% agarose gel electrophoresis, and the band at 1000bp was recovered by cutting as shown in b-diagram in FIG. 1. The recovery was performed according to the gel recovery kit of Beijing Optimala Biotech Ltd. Obtaining the ORF sequence of the PtrCHLP3 gene of the poplar. The nucleotide of the PtrCHLP3 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 12.

2. Construction of expression interfering plant expression vector pCAMBIA2301-PtrCHLP3-RNAi

Designing a forward fragment cloning primer PtrCHLP3-F1 (shown as a sequence in SEQ ID NO. 2) and PtrCHLP3-R1 (shown as a sequence in SEQ ID NO. 3) of a PtrCHLP3 gene sequence, and designing a reverse fragment cloning primer PtrCHLP3-F2 (shown as a sequence in SEQ ID NO. 4) and PtrCHLP3-R2 (shown as a sequence in SEQ ID NO. 5). The gene PtrCHLP3 of poplar shown in SEQ ID NO.1 is used as a template, and a PCR one-step directional cloning kit is used for cloning a target gene. 25 μ L reaction for forward fragment cloning:

SuperMix 12.5. mu.L, Primer PtrCHLP 3-F11. mu.L, Primer PtrCHLP 3-R11. mu.L, cDNA 2. mu.L, ddH2O 8.5.5. mu.L; 25 μ L reaction for reverse fragment cloning:

SuperMix 12.5. mu.L, Primer PtrCHLP 3-F21. mu.L, Primer PtrCHLP 3-R21. mu.L, cDNA 2. mu.L, ddH2O 8.5.5. mu.L; reaction procedure: pre-denaturation at 94 ℃ for 3min, 35cycles (94 ℃ for 30s, 62 ℃ for 30s, 72 ℃ for 1min), extension at 72 ℃ for 5 min; storing at 4 ℃. After the PCR reaction is finished, a forward target fragment and a reverse target fragment are obtained.

Plasmid pCAMBIA2301-PC was double digested with XbaI/SacI. The double enzyme system is (50 μ L): 10 XQuickCut Buffer 5 u L, SacI 2.5 u L, XbaI 2.5 u L, pCambia2301-PS 1 u g, ddH2O up to 50 u L; gently pipette, mix well, and incubate at 37 ℃ for 2.5 h. The linearized plasmid vector pCAMBIA2301-PC is recovered. The forward sequence, RTM sequence and reverse sequence of the partial fragment of PtrCHLP3 were sequentially recombined into PCAMbia2301-PS vector by using Clonexpress II recombination kit, and then transformed into E.coli competent cell DH5 alpha. Then, the positive clones transformed are selected by PCR identification and sequenced to obtain a recombinant plasmid of pCAMBIA2301-PC connected with the forward fragment and the reverse fragment, and a vector map of the pCAMBIA2301-PtrCHLP3-RNAi vector is shown in figure 2;

3. 84K poplar transformed by agrobacterium GV3101 mediated leaf disc method

Taking out 100 μ L of the agrobacterium-infected state from an ultralow temperature refrigerator, putting the agrobacterium-infected state on crushed ice for melting, respectively adding 5 μ L of suppression expression vector plasmid pCAMBIA2301-PtrCHLP3-RNAi into the crushed ice, and flicking and uniformly mixing; standing on ice for 5min, quick freezing with liquid nitrogen for 5min, water bath at 37 deg.C for 5min, and ice bath for 5 min. Adding 700. mu.L YEB culture medium, and culturing at 28 deg.C in shaking table at 200r/min for 2.5 h. Centrifuging at 6000r/min for 1min, collecting 100ul liquid culture medium, sucking, mixing, spreading on YEB solid culture medium containing 50mg/L Ka with coating rod, and culturing at 28 deg.C for 48 hr in inverted state. 6 monoclonal colonies were picked, and upstream primers 35S-F (SEQ ID NO.6) and downstream primers RTM-R (SEQ ID NO.7) were designed respectively based on the 35S fragment carried by the vector, and PCR was carried out using 1% agarose gel electrophoresis to detect the products as shown in c in FIG. 1. The samples were stored in glycerol with a final concentration of 20% for verification, frozen in liquid nitrogen and placed in a freezer at-80 ℃. And selecting a single colony for colony PCR, and inoculating the colony with a positive PCR result on a liquid culture medium containing kanamycin and rifampicin for shaking at 28 ℃ for 16 h. Centrifuging the activated bacterium liquid to obtain a precipitate, and resuspending the precipitate by using a liquid MS culture medium to obtain the agrobacterium tumefaciens bacterium liquid. When the concentration of the bacteria liquid measured by the spectrophotometer with the wavelength of 600nm is 0.6-0.8, the bacteria liquid can be used for infection conversion.

Taking the upper leaves of 84K poplar tissue culture seedlings, cutting into blocks of about 1 × 1cm by using scissors, using the main vein as a central axis, using a blade to cut a few knives on the main vein (a picture in figure 3), and then putting the leaves into the agrobacterium tumefaciens heavy suspension nutrient solution WPM to infect for about 10 min. Taking out the leaves, placing the leaves into a culture dish with filter paper, airing for several minutes, completely sucking residual bacterial liquid on the leaves by using a filter paper strip, transferring the leaves into WPM +100 mu M/L AS, and carrying out dark culture at 25 ℃ for 2 days. Transferring the infected leaves into WPM +0.2mg/L KT +1.0 mg/L2, 4-D +60mg/L Ka +250mg/L Cef +300mg/L TMT to induce the formation of callus, culturing at 25 ℃ in dark, and transferring the leaves into newly prepared WPM +0.2mg/L KT +1.0 mg/L2, 4-D +60mg/L Ka +250mg/L Cef +300mg/L TMT every 2 weeks. Cutting off when callus reaches rice size, transferring into WPM +0.02mg/L TDZ +60mg/L Ka +250mg/L Cef +300mg/L TMT to induce bud formation, culturing at 25 deg.C under illumination, transferring the callus into newly prepared WPM +0.02mg/L TDZ +60mg/L Ka +250mg/L Cef +300mg/L TMT every 2 weeks until more buds grow out of callus (figure 3, b). The shoots and the callus were inoculated into a hormone-free WPM +60mg/L Ka +250mg/L Cef together to induce the shoots to elongate. When the bud is elongated to 1cm, the bud is cut off, transferred into WPM +60mg/L Ka +250mg/L Cef to induce the bud to take root (c picture in figure 3), and cultured by illumination at 25 ℃ until the height of the plantlet is about 10 cm. The d-diagram in FIG. 3 shows the phenotype diagram of the control (WT), suppression expression (L1-L9) transgenic poplar subcultured for 30 days (note: the plants in FIGS. 3 and 4 correspond one to one). Selecting 84K poplar with well-developed root system, opening a cover, hardening seedlings in a culture room for 3d, transplanting the seedlings into a flowerpot, culturing the seedlings in an illumination incubator for 7d, and finally transferring the seedlings into a greenhouse for normal management. Untreated wild 84K poplar was used as control and the acclimatization method was the same as above. L1-L9 belong to the same transgenic plant, and are different in expression level of PtrCHLP3 gene.

4. Detection of transgenic lines

A part of the fragment schematic diagram of the interference expression vector inserted into the poplar genome is represented in a diagram in FIG. 4. And verifying the transgenic strain by adopting a PCR method. When the adventitious bud forms a complete plantlet, cutting partial leaves, extracting genome DNA (template) by using a CTAB method, and detecting a positive plant by using a gene specific primer. Designing a pair of specific primers 35S-F (shown as a sequence in SEQ ID NO.6) and RTM-R (shown as a sequence in SEQ ID NO.7) on CaMV35S and RTM respectively, taking an untransformed plant as a negative control (a wild plant), and carrying out primary PCR detection on a transgenic regeneration strain, wherein the PCR system is (25 mu L): 1.1 XT 3 Super PCR Mix 21. mu.L, 35S-F and RTM-R primers 1. mu.L each, DNA template 2. mu.L; the PCR procedure was pre-denaturation at 98 ℃ for 2min, 35cycles (98 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 30sec), extension at 72 ℃ for 2min, and storage at 4 ℃. The PCR products were detected by gel electrophoresis as shown in b of FIG. 4. the positive inhibitory expression lines of 9 (indicated by L1-L9) amplified bands of about 500bp, whereas the wild type plants did not.

And (4) verifying the transgenic plant strain by adopting a GUS staining method. Referring to the specification of a GUS staining kit of the Vietnamese organisms, cutting leaves, putting the cut leaves into a 1.5mL centrifuge tube, adding 1mL of GUS staining working solution, soaking the leaves, standing at 37 ℃ for 24h, and discarding the liquid. Adding 70% ethanol, soaking the leaves for decolorizing for 1-3h, 2-3 times, until the wild type 84K poplar leaves are white. The blue dots appearing in the leaves were observed as GUS expression sites. GUS staining observations are shown in the c-chart in FIG. 4, where 9 positive-suppression expression lines (L1-L9) were all blue, while the wild-type plants were white. And (3) selecting the leaves of the strain with PCR and GUS double detection as positive strains to extract RNA and perform reverse transcription to obtain cDNA, and then calculating the relative expression condition of the PtrCHLP3 of each strain by adopting fluorescent quantitative RT-PCR. A pair of primers PtrActin-F (shown in a sequence as SEQ ID NO. 10) and PtrActin-R (shown in a sequence as SEQ ID NO. 11) are designed by taking Actin as an internal reference, and a pair of specific primers CHLP3-F (shown in a sequence as SEQ ID NO. 8) and CHLP3-R (shown in a sequence as SEQ ID NO. 9) of the PtrCHLP3-ORF region are further designed. According to fluorescent quantitation kit (

Establishing an amplification system by Top Green qPCR SuperMix, wherein the amplification conditions are as follows: 30s at 94 ℃ in 40cycles (94 ℃ for 5sec, 60 ℃ for 15s, 72 ℃ for 10 s). Each sample is repeated for 3 times, the CT value of each sample is obtained according to data analysis, and the relative expression of the PtrCHLP3 of each transgenic line and the wild type is calculated. As shown in the d-chart in FIG. 4, the expression levels of the strains which inhibit the expression of the PtrCHLP3 gene are reduced compared with the Wild Type (WT), and particularly, the expression of PtrCHLP3 in the strains L3 and L4 is significantly inhibited. It was confirmed that the endogenous gene PtrCHLP3 had been transferred into 84KThe poplar genome is subjected to expression inhibition.

5. Statistics of measurement records of transgenic line phenotypes

Leaf color and chlorophyll analysis of transgenic positive lines (fig. 5): the leaf color of chlp3 and chlp4 turned significantly yellow compared to wild type (panel a in fig. 5). Meanwhile, chlorophyll content analysis shows that the contents of chlorophyll a, chlorophyll b and total chlorophyll in leaves of the transgenic plants are significantly lower than those of wild-type plants (b diagram in fig. 5).

Observations of the growth process of the transgenic positive lines showed that the height, ground diameter, growth, biomass (fresh weight), and internodes from 7 th to 8 th of the suppression-expressing plants were all reduced relative to the control (WT) plants (FIG. 6).

To further investigate the effect of PtrCHLP3 gene on poplar growth and development, we observed 35d of WT and transgenic poplar under normal conditions. Phenotypic analysis showed that the height of aerial parts and root length of transgenic poplar were significantly inhibited compared to wild-type poplar (panel a in fig. 6). With increasing growth time, both the height of the WT and the elongation of the stem were significantly higher than those of the transgenic plants (panels b and d in FIG. 6). In addition, the fresh weight of the root, stem and leaf of the transgenic poplar was significantly lower than that of the wild type, and was reduced by 30-75%, 42-84% and 46-82%, respectively (fig. 6, panel c). The stem diameter of the transgenic plants was significantly smaller compared to the wild type (fig. 6, panel e).

To further understand the effect of PtrCHLP3 on poplar stem thickness, we stained the cross-section between WT and chlp 8 th segments with toluidine blue and phloroglucinol-hcl (f-k panel in FIG. 6). The lignin (purple) in the cell wall was observed by m-chlorophenetriol-HCl staining. In wild type and transgenic plants, we observed no difference in lignin deposition in xylem, phloem fibers and myeloid cells (g, i and k panels in fig. 6). The width of phloem and xylem of transgenic poplar was significantly smaller than wild type. Furthermore, the proportion of transgenic xylem in the stem was significantly reduced compared to the wild type (panel I in fig. 6). However, there was no significant difference in bark ratio between the transgenic and wild-type stems. Consistent with previous results, quantitative analysis of the secondary xylem cell layer showed that the xylem cell layer of the transgenic plants was significantly smaller than WT (m-panels in fig. 6). These results indicate that inhibition of PtrCHLP3 expression can inhibit expansion of poplar xylem. In summary, PtrCHLP3 plays a very important role in regulating poplar growth.

The sequences of the primers used in the present invention are shown in Table 1:

TABLE 1 primers

In conclusion, the invention adopts transgenic technology to integrate a vector containing a partial fragment of the PtrCHLP3 gene into the poplar genome, and primarily directionally changes the growth rate and leaf color characteristics of the poplar. The PtrCHLP3 gene can slow down the growth of plant, reduce the plant height, thin the stem, yellow the leaf, etc. and provides new method for initial breeding of new poplar variety.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 9

actcacccac catgcacaaa 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cccattgagc acggtattgt 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tacgaccact ggcatacagg 20

<210> 12

<211> 470

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Ala Ser Ser Ile Val Phe Lys Ser Phe Thr Gly Leu Arg His Ser

1 5 10 15

Ser Gln Glu His Pro Lys Val Leu His Ser His Thr Asn Pro Ile Ser

20 25 30

Ser Phe Ser Tyr Ser Arg Phe Arg Ile Thr Ala Ser Lys Ser Ser Pro

35 40 45

Lys Leu Gln Asn Arg Asn Leu Arg Val Ala Val Ile Gly Gly Gly Pro

50 55 60

Ala Gly Gly Ala Ala Ala Glu Thr Leu Ala Lys Gly Gly Ile Glu Thr

65 70 75 80

Tyr Leu Ile Glu Arg Lys Leu Asp Asn Cys Lys Pro Cys Gly Gly Ala

85 90 95

Ile Pro Leu Cys Met Val Gly Glu Phe Asp Leu Pro Leu Asp Ile Ile

100 105 110

Asp Arg Arg Val Thr Lys Met Lys Met Ile Ser Pro Ser Asn Val Ala

115 120 125

Val Asp Ile Gly Arg Thr Leu Lys Pro His Glu Tyr Ile Gly Met Val

130 135 140

Arg Arg Glu Val Leu Asp Ala Tyr Leu Arg Glu Arg Ala Ser Thr Asn

145 150 155 160

Gly Ala Lys Val Ile Asn Gly Leu Phe Leu Lys Met Asp Ile Pro Lys

165 170 175

Lys Gly Ser Glu Asn Ile Asn Ser Pro Tyr Val Leu His Tyr Thr Glu

180 185 190

Tyr Asp Gly Lys Lys Gly Gly Thr Gly Glu Arg Lys Thr Leu Glu Val

195 200 205

Asp Val Val Ile Gly Ala Asp Gly Ala Asn Ser Arg Val Ala Lys Ser

210 215 220

Ile Asp Ala Gly Asp Tyr Asp Tyr Ala Ile Ala Phe Gln Glu Arg Ile

225 230 235 240

Lys Ile Pro Ser Asp Lys Met Val Tyr Tyr Glu Asn Leu Ala Glu Met

245 250 255

Tyr Val Gly Asp Asp Val Ser Pro Asp Phe Tyr Gly Trp Val Phe Pro

260 265 270

Lys Cys Asp His Val Ala Val Gly Thr Gly Thr Val Thr His Lys Gly

275 280 285

Asp Ile Lys Lys Phe Gln Leu Ala Thr Arg Asn Arg Ala Arg Asp Lys

290 295 300

Ile Leu Gly Gly Lys Ile Ile Arg Val Glu Ala His Pro Ile Pro Glu

305 310 315 320

His Pro Arg Pro Arg Arg Leu Ser Gly Arg Val Ala Leu Val Gly Asp

325 330 335

Ala Ala Gly Tyr Val Thr Lys Cys Ser Gly Glu Gly Ile Tyr Phe Ala

340 345 350

Ala Lys Ser Gly Arg Met Cys Ala Glu Ala Ile Val Glu Gly Ser Gly

355 360 365

Asn Gly Lys Arg Met Val Asp Glu Ser Asp Leu Arg Lys Tyr Leu Glu

370 375 380

Lys Trp Asp Lys Thr Tyr Trp Pro Thr Tyr Lys Val Leu Asp Val Leu

385 390 395 400

Gln Lys Val Phe Tyr Arg Ser Asn Pro Ala Arg Glu Ala Phe Val Glu

405 410 415

Met Cys Ala Asp Glu Tyr Val Gln Lys Met Thr Phe Asp Ser Tyr Leu

420 425 430

Tyr Lys Arg Val Val Pro Gly Asn Pro Leu Glu Asp Leu Lys Leu Ala

435 440 445

Val Asn Thr Ile Gly Ser Leu Val Arg Ala Asn Ala Leu Arg Arg Glu

450 455 460

Met Asp Lys Leu Ser Ala

465 470

Claims

1. A geranylgeraniol reductase, characterized by: the geranylgeraniol reductase is derived from poplar, and the amino acid sequence of the geranylgeraniol reductase is shown as SEQ ID NO. 12.

2. A biomaterial related to geranylgeraniol reductase as claimed in claim 1, wherein: the biological material is any one of the following 1) to 5):

1) a nucleic acid molecule encoding geranylgeraniol reductase;

2) an expression cassette comprising 1) the nucleic acid molecule;

3) a recombinant vector comprising 1) said nucleic acid molecule;

4) a recombinant microorganism comprising 1) said nucleic acid molecule;

5) a recombinant microorganism containing 3) the recombinant vector.

3. The biomaterial of claim 2, wherein: the nucleotide sequence of the nucleic acid molecule for coding geranylgeraniol reductase is shown as SEQ ID NO. 1.

4. Use of a geranylgeraniol reductase according to claim 1 or a biomaterial according to claim 2 or 3 for modulating the growth and development of poplar.

5. Use according to claim 4, characterized in that: the regulation and control of the growth and development of the poplar comprise the regulation and control of the growth rate and/or the leaf color of the poplar.

6. Use according to claim 4 or 5, characterized in that: the regulation comprises inhibiting the expression of the geranylgeraniol reductase or the biological material in the poplar so as to slow the growth rate of the poplar and/or yellow the leaf color.

7. A construction method of transgenic poplar is characterized in that: the method comprises the following steps:

(1) taking a gene sequence of a coding gene PtrCHLP3 of the poplar geranylgeraniol reductase shown in SEQ ID NO.1 as a template, and respectively amplifying a forward fragment and a reverse fragment by using a forward fragment cloning primer and a reverse fragment cloning primer;

8. The construction method according to claim 7, wherein: the forward fragment cloning primer in the step (1) isPtrCHLP3-F1AndPtrCHLP3-R1what is, what isThe reverse fragment cloning primer isPtrCHLP3-F2 andPtrCHLP3-R2; the forward fragment cloning primerPtrCHLP3-F1The sequence of (a) is shown as SEQ ID NO.2, and the forward fragment cloning primerPtrCHLP3-R1The sequence of (A) is shown as SEQ ID NO.3, the reverse fragment cloning primerPtrCHLP3-F2The sequence of (a) is shown as SEQ ID NO.4, and the reverse fragment cloning primerPtrCHLP3-R1The sequence of (A) is shown in SEQ ID NO. 5.

9. The construction method according to claim 7, wherein: in the step (3), the pCAMBIA2301-PtrCHLP3-RNAi expression vector is used for transforming poplar by a leaf disc method, and the method specifically comprises the following steps: transforming the pCAMBIA2301-PtrCHLP3-RNAi positive plasmid into expression strain competence, infecting poplar leaf disc by an infection method, and screening positive transgenic strains to obtain transgenic poplar.

10. The construction method according to claim 9, wherein: the expression strain is agrobacterium.