CN114717210B - Poplar geranylgeraniol reductase and encoding gene and application thereof - Google Patents

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 utilized to construct transgenic poplar, and the growth rate and leaf color characteristics of the poplar are primarily and directionally changed. The PtrCHLP3 gene inhibition expression can slow down plant growth, reduce plant height, thin stem, yellow leaf color and the like, and provides a new method for initially breeding Yang Shuxin varieties by utilizing a genetic engineering technology.

Description

Poplar geranylgeraniol reductase and encoding 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 and complete the fundamental process of capturing light energy in antenna systems by electron transfer driving reaction centers. Chlorophyll plays a vital role in photosynthetic organisms such as plants. Chlorophyll a is the primary electron donor of the PSI and PSII reaction centers. With the development of molecular biology and protein structure, key members involved in 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 a number of species, including photosynthetic bacteria, algae, tobacco, rice, peach, olive, and tomato. Chlorophyll and carotenoid content in chlp mutant of blue algae is reduced. Plants grow slowly after tobacco CHLP gene silencing, and leaves are in a pale or mottled phenotype. In rice, the chlp mutant has yellow-green leaves, which affects plant growth. In poplar, ptrCHLP3 gene plays an important role in regulating growth rate, photosynthetic rate, leaf color and the like of poplar. Therefore, screening and cloning of PtrCHLP3 gene, genetic transformation using PtrCHLP3 gene, is one of the important ways to improve new plant variety and create colored-leaf plants.

The color leaf tree species are important material foundations for building park cities. However, the current color-leaf garden tree variety is single and scarce, and can not meet the requirement of urban landscaping, so that the cultivation of new color-leaf tree varieties has become an important target for garden tree breeding. Therefore, a rapid and directional improved molecular breeding technology is needed to make up for the defects of the traditional garden tree breeding technology, and to efficiently select new varieties of colored-leaf trees so as to meet the requirements of urban park construction. Existing molecular breeding for color transformation mainly focuses on transformation of flower colors of herbs, and molecular breeding of color-leaved trees is almost blank at home and abroad. Because the key genes and the regulation and control mechanisms of the color formation of the color tree are not clear, the defects and the bottlenecks greatly block the molecular breeding process of the color tree, and therefore, reports of modifying the color of the tree through molecular biology means and breeding new varieties of the color tree are few. At present, the research on the PtrCHLP3 gene of poplar is not reported yet, but the technology for improving new poplar varieties and creating colored-leaf plants by using the PtrCHLP3 gene is lacking. If the expression of PtrCHLP3 gene can be regulated and controlled by the technology of genetic engineering, the growth rate and leaf color of poplar can be improved gradually, the application of the PtrCHLP3 gene in production can be promoted greatly, and the PtrCHLP3 gene has very important practical significance for the molecular breeding research of forest trees and the ecological civilization construction of forestry.

Disclosure of Invention

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

in a first aspect, the invention provides a geranylgeraniol reductase from poplar, having the amino acid sequence shown in SEQ ID NO. 12.

In a second aspect, the present invention also provides a biomaterial associated with the geranylgeraniol reductase, any one of the following 1) to 5):

1) A nucleic acid molecule encoding a geranylgeraniol reductase;

2) An expression cassette comprising 1) said nucleic acid molecule;

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

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

5) Recombinant microorganism comprising 3) said recombinant vector.

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

In a third aspect, the invention provides the use of said geranylgeraniol reductase or said biomaterial for modulating the growth and development of poplar.

Further, the controlling poplar growth comprises controlling poplar growth rate and/or leaf color.

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

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

(1) The gene sequence of the gene PtrCHLP3 of the poplar geranylgeraniol reductase shown in SEQ ID No.1 is used as a template, and a forward fragment cloning primer and a reverse fragment cloning primer are used for amplifying the forward fragment and the reverse fragment respectively;

(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 part of PtrCHLP3 fragment into the PCAMBIA2301-PS vector to obtain pCAMBIA2301-PtrCHLP3-RNAi positive plasmid;

(3) The pCAMBIA2301-PtrCHLP3-RNAi positive plasmid was used to transform poplar by 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 PtrCHLP3-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 pCAMBIA2301-PtrCHLP3-RNAi expression vector is transformed into poplar by adopting a leaf disc method, and the method specifically comprises the following steps: and transforming pCAMBIA2301-PtrCHLP3-RNAi positive plasmid into an expression strain competence, infecting a poplar leaf disc by using an infection method, and screening a positive transgenic strain to obtain the transgenic poplar.

Preferably, the expression strain is agrobacterium.

The invention has the beneficial effects that:

the invention provides a novel application of a poplar PtrCHLP3 gene in regulating and controlling the growth and development of poplar, in particular regulating and controlling the growth speed, photosynthetic rate and leaf color of poplar. And then, the transgenic technology is adopted to integrate the vector containing partial fragments of PtrCHLP3 genes into the poplar genome, and the growth rate and leaf color characteristics of the poplar are primarily and directionally changed. The PtrCHLP3 gene inhibition expression can slow down plant growth, reduce plant height, thin stem, yellow leaf color and the like, and provides a new method for initially breeding Yang Shuxin varieties by utilizing a genetic engineering technology.

Drawings

FIG. 1 is a diagram showing the correlation detection electrophoresis of a PtrCHLP 3-repressed expression vector constructed in example 1 of the present invention; (a) total RNA electrophoresis detection; (b) full-length electrophoretogram of the target fragment; 1 to 4: a full-length fragment of the target gene PtrCHLP 3; m is Mark 2000; (c) PCR verification of Agrobacterium transformation electrophoresis pattern M: mark 2000;1 to 6: PCR verification of pCAMBIA2301-PtrCHLP3-RNAi recombinant plasmid;

FIG. 2 is a vector map of the expression-repressing 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 PtrCHLP 3-transgenic poplar in example 1 of the present invention; wherein a: callus; b: regenerating transgenic bud clusters by leaf discs; c: regenerated resistant transgenic plantlets; d: phenotype map of control (WT), repressed expression (L1-L9) transgenic tender plants subcultured for 30 days;

FIG. 4 is a diagram showing different detection modes of poplar transformed with PtrCHLP3 gene in example 1 of the present invention; (a) Constructing a schematic fragment of a plant expression vector pCAMBIA2301-PtrCHLP3-RNAi for inhibiting expression; (b) detecting an electrophoresis pattern by transferring PtrCHLP3 gene poplar specific primers; m: mark 2000; PC: p2301-PnCHLP-RNAi positive control vector; NC: a negative control; WT: wild type; L1-L9: ptrCHLP3 represses expression of poplar transgenic lines; (c) histochemical GUS staining patterns of different transgenic plants; WT: wild type; L1-L9: ptrCHLP3 represses expression of poplar transgenic lines; (d) Analyzing the relative expression quantity of PtrCHLP3 genes of different transgenic lines; WT: wild type; L1-L9: ptrCHLP3 represses expression of poplar transgenic lines;

FIG. 5 is a phenotype chart of a PtrCHLP3 gene-transferred poplar in example 1 of the present invention and chlorophyll content; (a) poplar leaf top view; WT: wild type; chlp-3/4: ptrCHLP3 represses expression of poplar transgenic lines; (b) poplar chlorophyll content; chla: chlorophyll a; chlb: chlorophyll b; caro: carotenoids;

FIG. 6 is a 35 day seedling phenotype statistical analysis of control (WT), inhibition expression (chlp-3/4) positive lines in example 1 of the present invention. (a): a representation map; (b): relationship between plant height and days; (c): fresh weight of different parts; (d): stem growth; (e): a ground diameter; (f) - (k): 8, a section stem cross section staining chart; (i) - (m): and 8, counting stem cross section data.

Detailed Description

In the description of the present invention, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.

Example 1

1. Acquisition of PtrCHLP3 Gene

The total RNA extraction material is young leaves of 84K poplar, the young leaves are fully ground by adopting liquid nitrogen, the extraction is carried out by referring to the RNA extraction kit instruction book of RNAprep Pure Plant Kit of Alrick biological company in Hunan province, an RA-10 nuclease agent is matched with the spray experiment instrument and the operation table board in the experiment process, and the RNase remained on the surface is removed. RNA concentration, OD260/OD280, and OD260/OD230 values were measured using a Thermo Nanodrop 2000 nucleic acid/protein quantifier, and the absorbance peak at 260nm of the sample was observed. RNA integrity was checked by electrophoresis on 150V,20min,1% agarose gel and the results are shown in FIG. 1, panel a. cDNA Synthesis reference Hunan Airake BioCo

All-in-One First-Strand cDNA Synthesis SuperMix for PCR kit instructions. The nucleotide sequence of the poplar CHLP3 gene is obtained according to the populus database populus genome of popkenie, primers PtrCHLP3-F1 (the sequence is shown as SEQ ID NO. 2) and PtrCHLP3-R1 (the sequence is shown as SEQ ID NO. 3) are designed by using Primer 5.0, and the cDNA synthesized in the previous step is used as a template to amplify the whole length of the gene by PCR. 25. Mu.L of reaction system: />

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, ddH ₂ O8.5 μl; the reaction procedure: pre-denatured at 94℃for 3min,35cycles (94℃30s,62℃30s,72℃1 min), extension at 72℃for 5min; preserving at 4 ℃. After the completion of the PCR reaction, the band at 1000bp was cut and recovered by 1% agarose gel electrophoresis as shown in FIG. 1 b. The recovery was performed according to the glue recovery kit of the biological limited of the family beijing. The ORF sequence of the PtrCHLP3 gene of poplar is obtained. The nucleotide of PtrCHLP3 gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 12.

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

Design of PtrCHLP3 radicalThe forward fragment cloning primer PtrCHLP3-F1 (the sequence is shown as SEQ ID NO. 2) and PtrCHLP3-R1 (the sequence is shown as SEQ ID NO. 3), the reverse fragment cloning primer PtrCHLP3-F2 (the sequence is shown as SEQ ID NO. 4) and PtrCHLP3-R2 (the sequence is shown as SEQ ID NO. 5). The target gene is cloned by using a PCR one-step directional cloning kit by taking the poplar PtrCHLP3 gene shown in SEQ ID NO.1 as a template. 25. Mu.L reaction system for forward fragment cloning:

supermix 12.5. Mu.L, primer PtrCHLP3-F1 1. Mu.L, primer PtrCHLP3-R1 1. Mu.L, cDNA 2. Mu.L, ddH2O 8.5. Mu.L; reverse fragment clone 25 μl reaction system: />

Supermix 12.5. Mu.L, primer PtrCHLP3-F2 1. Mu.L, primer PtrCHLP3-R2 1. Mu.L, cDNA 2. Mu.L, ddH2O 8.5. Mu.L; the reaction procedure: pre-denatured at 94℃for 3min,35cycles (94℃30s,62℃30s,72℃1 min), extension at 72℃for 5min; preserving at 4 ℃. After the PCR reaction is finished, the forward target fragment and the reverse target fragment are obtained.

Plasmid pCAMBIA2301-PC was digested with XbaI/SacI. The double cleavage system was (50. Mu.L): 10 XQuickCut Buffer 5. Mu.L, sacI 2.5. Mu.L, xbaI 2.5. Mu.L, pCambia2301-PS 1. Mu.g, ddH2O up to 50. Mu.L; gently pipetting and mixing well and incubating at 37℃for 2.5h. The linearized plasmid vector pCAMBIA2301-PC was recovered. And (3) sequentially recombining the forward sequence of the PtrCHLP3 partial fragment, the RTM sequence and the reverse sequence of the partial fragment into a PCAMbiA2301-PS vector by using a Clonexpress II recombination kit, and then converting into an escherichia coli competent cell DH5 alpha. Then, the positive clones transformed are identified and selected through PCR and sequenced to obtain recombinant plasmids of pCAMBIA2301-PC connected with the forward fragment and the reverse fragment, and the pCAMBIA2301-PtrCHLP3-RNAi vector is obtained, and the vector map is shown in figure 2;

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

Taking out 100 mu L of agrobacterium competent from an ultralow temperature refrigerator, putting the agrobacterium competent onto crushed ice for melting, respectively adding 5 mu L of inhibition expression vector plasmids pCAMBIA2301-PtrCHLP3-RNAi into the crushed ice, and mixing the crushed ice with light bullets; sequentially standing on ice for 5min, quick-freezing with liquid nitrogen for 5min, water-bathing at 37deg.C for 5min, and ice-bathing for 5min. mu.L of YEB medium was added and incubated at 28℃for 2.5h at 200r/min in a shaker. Centrifuging at 6000r/min for 1min, collecting 100ul of liquid culture medium, sucking, stirring, mixing, coating on YEB solid culture medium containing 50mg/L Ka with a coating rod, and culturing at 28deg.C for 48 hr in an inverted manner. 6 monoclonal colonies were picked, the upstream primer 35S-F (SEQ ID NO. 6) was designed based on the vector-carried 35S fragment, the vector RTM fragment was designed to the downstream primer RTM-R (SEQ ID NO. 7) for PCR reaction, and the products were detected by 1% agarose gel electrophoresis as shown in FIG. 1, panel c. The verification was positive in ensuring that the glycerol was stored at a final concentration of 20%, and the liquid nitrogen was frozen and placed in a-80 ℃ freezer. Single colonies were picked for colony PCR, and colonies positive in PCR result were inoculated on a liquid medium containing kanamycin and rifampicin, and shaken at 28℃for 16 hours. Centrifuging the activated bacterial liquid to obtain a precipitate, and re-suspending the precipitate with a liquid MS culture medium to obtain the agrobacterium bacterial liquid. The method can be used for infection conversion when the concentration of the bacterial liquid is measured to be 0.6-0.8 by using a spectrophotometer with a wavelength of 600 nm.

The upper leaf blade of 84K poplar tissue culture seedling is cut into blocks of about 1X 1cm by scissors, the main vein is used as a central axis, a cutter is used for cutting a few cutters on the main vein (a diagram in fig. 3), and then the upper leaf blade is placed into agrobacterium resuspension nutrient solution WPM for infection for about 10 min. Taking out the leaf, airing in a culture dish with filter paper for several minutes, thoroughly sucking the residual bacterial liquid on the leaf by using a filter paper strip, transferring the leaf into WPM+100 mu M/L AS, and culturing in dark at 25 ℃ for 2d. Infection leaves are transferred into WPM+0.2mg/L KT+1.0mg/L2, 4-D+60mg/L Ka+250mg/L Cef+300mg/L TMT to induce callus formation, dark culture is carried out at 25 ℃, and the leaves are transferred 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 the callus until the grain size reaches the grain size, transferring the callus into WPM+0.02mg/L TDZ+60mg/L Ka+250mg/L Cef+300mg/L TMT to induce bud formation, culturing at 25 ℃ by illumination, and 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 on the callus (b graph in 3). The shoots and calli were inoculated together into hormone-free WPM+60mg/L Ka+250mg/L Cef and the shoots were induced to undergo elongation growth. When the buds are elongated to 1cm, the buds are cut off and transferred into WPM+60mg/L Ka+250mg/L Cef to induce the buds to root (figure 3, c), and the buds are cultivated by illumination at 25 ℃ until the height of the seedlings is about 10cm. Panel d in FIG. 3 shows a phenotype of control (WT), repressed expression (L1-L9) transgene for Yang Shuji passages cut for 30 days (note: one-to-one correspondence of plants in FIGS. 3 and 4). 84K poplar with well developed root system is selected, the poplar is covered in a culture room to smelt seedlings for 3d, then is transplanted into a flowerpot, is cultured in an illumination incubator for 7d, and finally is transferred into a greenhouse for normal management. The untreated wild 84K poplar was used as a control, and the seedling hardening was performed in the same manner as above. L1 to L9 belong to the same transgenic plant, with the difference that the expression level of PtrCHLP3 gene is different.

4. Detection of transgenic lines

FIG. 4, panel a, is a schematic representation of a portion of a fragment representing an interfering expression vector inserted into the poplar genome. And (5) verifying the transgenic strain by adopting a PCR method. When the adventitious bud forms a complete plantlet, part of the leaf is cut, genomic DNA (template) is extracted by using a CTAB method, and a positive plant is detected by using a gene specific primer. A pair of specific primers 35S-F (sequence shown as SEQ ID NO. 6) and RTM-R (sequence shown as SEQ ID NO. 7) are respectively designed on CaMV35S and RTM, untransformed plants are used as negative controls (wild type plants), and primary PCR detection is carried out on transgenic regenerated strains, wherein the PCR system is (25 mu L): 1.1X1.3 Super PCR Mix 21. Mu.L, 35S-F and RTM-R primers each 1. Mu.L, DNA template 2. Mu.L; PCR procedure was 98℃for pre-denaturation of 2min,35cycles (98℃for 10s,60℃for 10s,72℃for 30 sec), 72℃for 2min and 4℃for storage. As shown in panel b of FIG. 4, 9 positive inhibition expression lines (represented by L1-L9) can be amplified by gel electrophoresis of the PCR products to form a band with a size of about 500bp, while the wild-type plants do not amplify the band.

And verifying the transgenic strain by adopting a GUS staining method. Referring to GUS staining kit instruction of Vaccinium macrocarpon, cutting the leaves, placing into a 1.5mL centrifuge tube, adding 1mL GUS staining working solution to soak the leaves, standing at 37deg.C for 24h, and discarding the liquid. Adding 70% ethanol to soak leaves for decoloring for 1-3h and 2-3 times until the wild 84K poplar leaves are white. The blue dots appearing in the leaf were observed to be GUS expression sites. GUS staining observations are shown in FIG. 4, panel c, where 9 positive inhibition expression lines (L1-L9) were allBlue, while wild type plants are white. And selecting leaves of positive strains with PCR and GUS double detection, extracting RNA, reversely transcribing the RNA into cDNA, and calculating the relative expression condition of PtrCHLP3 of each strain by adopting fluorescent quantitative RT-PCR. A pair of primers PtrAlctin-F (with the sequence shown as SEQ ID NO. 10) and PtrAlctin-R (with the sequence shown as SEQ ID NO. 11) are designed by taking an action as an internal reference, and a pair of specific primers CHLP3-F (with the sequence shown as SEQ ID NO. 8) and CHLP3-R (with the sequence shown as SEQ ID NO. 9) of PtrCHLP3-ORF region are designed. According to the fluorescent quantitative kit

Top Green qPCR SuperMix amplification system was established, amplification conditions: 94℃30s,40cycles (94℃5sec,60℃15s,72℃10 s). Each sample was repeated 3 times, and the CT values of each sample were obtained by data analysis, and the relative expression of each transgenic line and wild-type PtrCHLP3 was calculated. As shown in the graph d of FIG. 4, the amount of expression of the PtrCHLP 3-inhibiting expression strain was reduced as compared with the wild-type strain (WT), and particularly, the PtrCHLP3 expression was significantly inhibited in the strains L3 and L4. It was confirmed that the endogenous gene PtrCHLP3 had been transferred into the 84K poplar genome and the result of repression of expression was obtained.

5. Measurement record statistics of transgenic line phenotypes

Leaf color and chlorophyll analysis for transgenic positive lines (fig. 5): leaf color of chlp3 and chlp4 turned significantly yellow compared to wild type (panel a in fig. 5). Meanwhile, chlorophyll content analysis showed that chlorophyll a, chlorophyll b, and total chlorophyll content in leaves of transgenic plants were significantly lower than that of wild type plants (panel b in fig. 5).

Observations of the growth process of transgenic positive lines showed that the inhibition of expressed plant height, ground diameter, growth, biomass (fresh weight), and internodes 7-8 were all reduced relative to control (WT) plants (fig. 6).

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

To further understand the effect of PtrCHLP3 on poplar stem thickness, we stained the cross-section between section 8 of WT and chlp plants with toluidine blue and phloroglucinol-hcl (f-k plot in FIG. 6). Lignin (red violet) in the cell wall was observed by phloroglucinol-HCl staining. In wild-type and transgenic plants, we observed no difference in lignin deposition in xylem, phloem fiber and medulla cells (g, i and k panels in fig. 6). The width of phloem and xylem of transgenic poplar is significantly smaller than that of 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 plot in fig. 6). These results indicate that inhibiting expression of PtrCHLP3 can inhibit expansion of poplar xylem. Taken together, 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 summary, the invention adopts transgenic technology to integrate the vector containing partial segment of PtrCHLP3 gene into poplar genome, and primarily changes the growth rate and leaf color characteristics of poplar. The PtrCHLP3 gene inhibition expression can slow down plant growth, reduce plant height, thin stem, yellow leaf color and the like, and provides a new method for initially breeding Yang Shuxin varieties by utilizing a genetic engineering technology.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

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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 (9)

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. 14.

2. A biological material associated with the geranylgeraniol reductase of claim 1, wherein: the biological material is any one of the following 1) to 5):

1) A nucleic acid molecule encoding a geranylgeraniol reductase;

2) An expression cassette comprising 1) said nucleic acid molecule;

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

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

5) Recombinant microorganism comprising 3) said recombinant vector.

3. The biomaterial according to claim 2, characterized in that: the nucleotide sequence of the nucleic acid molecule for encoding the geranylgeraniol reductase is shown as SEQ ID NO. 1.

4. Use of a geranylgeraniol reductase of claim 1 or a biomaterial of claim 2 or 3 for modulating poplar growth; the regulation of the growth and development of poplar is to regulate the growth rate and/or leaf color of poplar.

5. The use according to claim 4, characterized in that: the modulation is to inhibit expression of the geranylgeraniol reductase or the biological material in the poplar to thereby slow down the growth rate of the poplar and/or yellow leaf color.

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

(1) Poplar geranylgeraniol reductase coding gene shown in SEQ ID NO.1PtrCHLP3The gene sequence of (2) is used as a template, and a forward fragment cloning primer and a reverse fragment cloning primer are respectively utilized to amplify a forward fragment and a reverse fragment;

(2) PCAMBIA2301-PS is taken as a framework vector, the PCAMBIA2301-PS vector is subjected to double enzyme digestion, and thenPtrCHLP3The forward sequence of the partial fragment, the RTM sequence and the reverse sequence of the partial fragment are recombined into a PCAMBIA2301-PS vector in sequence to obtain a pCAMBIA2301-PtrCHLP3-RNAi positive plasmid;

(3) The pCAMBIA2301-PtrCHLP3-RNAi positive plasmid was used to transform poplar by leaf disc method.

7. The construction method according to claim 6, wherein: the forward fragment cloning primer in the step (1) isPtrCHLP3-F1AndPtrCHLP3-R1the reverse fragment cloning primer isPtrCHLP3-F2 andPtrCHLP3-R2; the forward fragment cloning primerPtrCHLP3-F1The sequence of the forward fragment cloning primer is shown as SEQ ID NO.2PtrCHLP3-R1The sequence of the reverse fragment clone primer is shown as SEQ ID NO.3PtrCHLP3-F2The sequence of the reverse fragment clone primer is shown as SEQ ID NO.4PtrCHLP3-R1The sequence of (2) is shown as SEQ ID NO. 5.

8. The construction method according to claim 6, wherein: in the step (3), the pCAMBIA2301-PtrCHLP3-RNAi expression vector is transformed into poplar by adopting a leaf disc method, and the method specifically comprises the following steps: and transforming pCAMBIA2301-PtrCHLP3-RNAi positive plasmid into an expression strain competence, infecting a poplar leaf disc by using an infection method, and screening a positive transgenic strain to obtain the transgenic poplar.

9. The method of construction according to claim 8, wherein: the expression strain is agrobacterium.

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