LU502442B1 - Sugar transporter gene for affecting wood formation in poplar and application thereof - Google Patents

Abstract

The invention provides a sugar transporter gene for affecting wood formation and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the sugar transporter gene is shown in SEQ ID NO: 1, and the amino acid sequence of encoded protein is shown in SEQ ID NO: 2. According to the invention, the sugar transporter gene (PagSWEET17a) was cloned from 84K. The over-expression vector pMDC32-PagSWEET17a was constructed and transferred into poplar mediated by A. tumefaciens. Compared with those of non-transgenic control 84K, the xylem area of transgenic poplars overexpressing PagSWEET17a was wider, and the secondary cell wall of the xylem fibers and vessels was thicker. PagSWEET17a is involved in the transportation of sugar to xylem, which affects the formation of wood and has important application value in the field of forest genetic engineering.

Description

DESCRIPTION LU502442

SUGAR TRANSPORTER GENE FOR AFFECTING WOOD FORMATION IN POPLAR AND APPLICATION THEREOF

TECHNICAL FIELD The invention relates to the technical field of plant genetic engineering, and in particular to a sugar transporter gene for affecting wood formation in poplar and application thereof.

BACKGROUND Wood is the most important renewable resource on the earth and the main raw material of pulp and paper making. Genetic improvement on the existing tree species is an important way to improve the timber yield. Poplar (Populus L.) is an economic tree species widely distributed in the world, and is also widely used in intensive cultivation because of its fast growth, strong adaptability and easy genetic manipulation. Poplar has always been an important tree species to obtain raw materials for papermaking. With the development of genetics, genomics and molecular biology technology in recent years, poplar has become a model plant for studying wood formation and other biological processes in trees. Therefore, it is of great theoretical significance and has application value to study molecular mechanism underlying wood formation in poplar.

Wood is the lignified tissue formed through the secondary growth of woody plants. It generally includes the secondary vascular tissues produced by the near-axial division and differentiation of vascular cambium. The radial growth of trees is the result of the periodic activity of secondary vascular cambium, which involves many biological processes such as cell division, cell expansion, secondary cell wall formation and programmed cell death. In the process of wood formation, it is necessary to continuously obtain carbon sources from photosynthetic tissues to meet the development of xylem. Therefore, the efficiency of carbon assimilation and transportation in tree determine the yield and quality of wood. In this process, plant sugar transporters are responsible for the transmembrane transport of sugar, and play an important role in the distribution of carbon sources to xylem, directly affecting wood formation in trees. Therefore, studying the role of sugar transporters in woody plants is helpful to reveal the molecular mechanism of wood formation, thus providing new ideas for molecular improvemeht/502442 of wood formation in forest trees.

SWEET; (Sugars Will Eventually be Exported Transporters) are sugar transporters found in recent years. Studies on herbs found that SWEETS influent many biological processes of plants, including vegetative growth, reproductive growth, senescence, stress resistance, interaction between plants and microorganisms, by controlling sugar transportation. At present, the research on SWEETs mainly focuses on herbaceous plants. Different from herbaceous plants, perennial woody plants have special development processes such as secondary growth, and their carbon transport mechanism is obviously different from that of herbaceous plants. However, at present, the research on carbon transport of trees, especially the radial transport of stems, is still very limited, and the molecular mechanism of carbon accumulation in xylem is still unclear. In view of the importance of SWEET in plant growth and development, the present invention studies its role in wood formation.

SUMMARY The purpose of the present invention is to provide a sugar transporter gene for affecting wood formation in poplar and application thereof. Overepression of this gene promote the development of xylem by widenning the xylem area and thickenning the secondary cell wall. It has important application value in the field of forest genetic engineering.

To achieve the above purpose, the present invention provides the following solutions: The invention provides a sugar transporter gene (PagSWEET17a) for affecting wood formation in poplar, and the nucleotide sequence of which is shown in SEQ ID NO: 1.

The invention also provides a protein encoded by the sugar transporter gene, and the amino acid sequence of which is shown in SEQ ID NO: 2.

The invention also provides a pair of primers for amplifying the sugar transporter gene, which comprises an upstream primer shown in SEQ ID NO: 4 and a downstream primer shown in SEQ ID NO: 5.

The invention also provides a recombinant vector comprising the sugar transporter gene, and its nucleotide sequence is shown in SEQ ID NO: 3.

The invention also provides a recombinant bacterium comprising the recombinant vector.

The invention also provides a method for constructing the recombinant vector, whi&h/502442 comprises the following steps: (1) PCR amplification by the primer set of claim 3 using with poplar cDNA as a template to obtain the sugar transporter gene of claim 1; (2) Carrying out a recombination reaction on the sugar transporter gene and an entry vector to obtain a recombinant entry vector; (3) Recombining the recombinant entry vector into a plant expression vector, and performing LR reaction to introduce the sugar transporter gene into the plant expression vector, assembling the constitutive promoter p35S at the 5' end and the strong terminator NOS at the 3' end of the sugar transporter gene to obtain the recombinant vector.

The invention also provides the application of the sugar transporter gene, its protein, the primers using for PCR amplification, recombinant vector, and the recombinant bacteria in regulating the growth and development of poplar.

Further, the application is specifically in regulating and controlling wood formation.

Further, the regulation of wood formation includes promoting the widening of xylem and thickening of secondary cell wall in fibers and vessels.

The invention discloses the following technical effects: According to the invention, the gene PagSWEET17a was cloned from 84k (P alba x P. glandulosa). An over-expression vector pPMDC32PagSWEKET17a was constructed. In the vector, the gene is located behind the promoter p35S. Under the drive of the promoter p35S, PagSWEET17a can be efficiently expressed in poplar, thereby influent the development of xylem in poplar. PagSWEET17a is involved in the transportation of sugar from leave to xylem, which affects the formation of wood formation in poplar. Compared with the prior art, the present invention present a gene which can widen the area of xylem and thicken the secondary cell wall of fibers and vessels by overexpressing PagSWKET17a in poplar. It has important application value in the field of forest genetic engineering.

BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments or the technical solutions involved in the present invention, the following description will briefly introduce the figures in the embodiments. The following figures are only some of embodiments of the present invention. For those of ordinary skill in the art, other figures can be obtained according to these figures without ats/502442 creative effort.

Fig. 1 shows a comparison on stem diameters of non-transgenic 84K and transgenic poplars overexpressing PagSWEET17a in Example 4 of the present invention; Fig. 2 shows cross-sections of non-transgenic 84K and transgenic poplars overexpressing PagSWEET17a in Example 4 of the present invention; Fig. 3 shows secondary cell walls in xylem of non-transgenic 84K and transgenic poplars overexpressing PagSWEET 17a in Example 4 of the present invention.

DESCRIPTION OF THE INVENTION The following is the detailed description of the exemplary embodiments in the present invention. This detailed description should not be taken as a limitation of the present invention, but should be understood as a more detailed description of some aspects, characteristics and embodiments of the present invention.

It should be understood that the terms mentioned in the present invention are only used to describe specific embodiments, and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Every smaller range between stated values or the intermediate value within the stated range is also included in the present invention.

Unless otherwise stated, all technical and scientific terms used herein have the same meanings commonly understood by those of ordinary skill in the field. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.

Without departing from the scope of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present specification. It is also obvious that other embodiments can be obtained from the description of the present invention by the skilled person. The description and embodiment of thé/502442 invention are only exemplary.

As used in this paper, the terms "comprising", "including", "having" and "containing" are all open terms, meaning including but not limited to.

According to the invention, PagSWEET17a in PagSWEETs family is highly expressed in phloem and xylem, and it may play an important role in wood formation. Studying the function of this gene in wood formation is of great significance for further revealing the molecular mechanism underlying wood formation. And, it will facilitate the breeding of new forest varieties with significantly improved wood properties through molecular breeding.

Example 1 PCR amplification of PagSWEET17a The total RNA was extracted from stem segments of 84k (P albaxP. glandulos) using RNeasy Plant mini Kit (Tiangen Biochemical Technology Beijing Co., Ltd). The first strand of cDNA was synthesized using Superscripag III first strand synthesis system (Invitrogen). With reference to the published genome sequence of P. tomentosa, primers were designed by Primer3 software, and the full-length gene, including start codon and stop codon, was amplified (GATEWAY linker was introduced into the primers).

The forward primer and reverse primer sequences used for PagSWKET17a amplification are as follows: Forward primer: 5’-GGGGACAACTTTGTACAAAAAAGTTGGAAT GGAAAGTCTCAGTTTGAT -3’ (SEQ ID NO: 4) Reverse primer: 5S’-GGCGGCCGCACAACTTTGTACAAGAAAGTT GGGTACTATGCTTCATTATTTTCTC-3’ (SEQ ID NO: 5) The PCR reaction system is as follows: TaKaRa high-fidelity amplification enzyme PrimeSTAR 12.5 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM)1 ul, template (cDNA) 1 ul, sterile ddH>O supplemented to 25 ul. Reaction procedure: pre-denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 53°C for 30 s and extension at 72°C for 1 min. Final extension was at 72°C for 7 min.

The full-length cDNA sequence of the obtained gene is 723bp, named as PagSWEET 17a, and the sequence is shown in SEQ ID NO: 1, and the coded protein sequence is shown in SEQ ID NO: 2.

Example 2 Construction of a recombinant vector for the overexpression of PagSWEET 1 Td)502442 in poplar The vector overexpressing PagSWEETI7a was created by Gateway recombinant cloning technology. The coding sequence of PagSWEETI7a was amplified from cDNA of 84K using specific primer of PagSWEET17a (the primers in Example 1), and recombined into entry vector pDNOR222.1 (SEQ ID NO: 6). The reaction system was as follows: 80 ng of PCR product; 0.4 ul of PDNOR222 1; 0.6 ul of BP Clonase II enzyme mix; sterile ddH2O supplemented to 5 ul. The reaction procedure was set as 25°C for 5 h to obtain entry clones.

The construction of entry clone was confirmed by sequencing. Then, the entry vector carrying PagSWEET17a was linearized by restriction enzyme Mlu I, and recombined into a binary vector pMDC32 (sequence shown in SEQ ID NO: 7). The LR reaction was carried out, and the reaction system is as follows: 50ng of linearized entry clone; 75ng of purified destination vector; 0.6 ul of LR Clonasell enzyme mix; TE buffer (pH 8.0) was made up to 5 ul. The reaction was carried out at 25°C for 5 h. After LR reaction, PagSWEET17a was introduced into pMDC32, and a constitutive promoter p35S was assembled at the 5' end of PagSWEETI7a, which could efficiently drive its expression in poplar. The transcription of PagSWEET17a can be effectively terminated by assembling a strong terminator NOS at the 3' end of the gene, thus obtaining the 35S: PagSWEET17a overexpression vector.

Hygromycin B phosphotransferase (HPT) was assembled on the vector plasmid, and was used as a screening marker for transgenic poplars. The LB and RB sequences were assembled in the vector plasmid, and promoted the integration of PagSWKET17a and HPT into poplar chromosome. Through PCR detection and sequencing verification, it was confirmed that the recombinant vector overexpressing PagSWEET17a was successfully constructed and named pMDC32-PagSWEET17a (the sequence is shown in SEQ ID NO: 3). Driven by the promoter p35S, PagSWEET17a can be highly expressed in poplar.

Example 3 Preparation of transgenic poplar overexpressing PagSWEET17a Using an electric shock method, the constructed vector pMDC32-PagSWEETI7a was transferred into Agrobacterium tumefaciens GV3101. The transformation was carried out according to the following steps. The tissue culture seedlings of 84 K used for genetic transformation were cultured at 23-25°C, 16/8 h (day/night) and 50 uMm-25s-1. A. tumefaciens containing pMDC32-PagSWEET17a infected 84K leaf disc at OD600=0.6-0.8. The infectéd/502442 discs were incubated for a total of 3 days in the dark at 22+2°C on adventitious bud induction medium (SIM, i.e, Murashige-Skoog (MS) minimal medium plus 0.5 mg/L 6-Benzyl Amino Purine (6-BA) and 0.05 mg/L Naphthalene Acetic Acid (NAA)). Then, the cultured leaf discs were transferred to SIM containing 3mg/L hygromycin B and 200 mg/L Timentin. The resistant adventitious buds were induced and screened under the conditions of 23-25°C, 16/8 h (day/night) and 50 pMm-2s-1 light intensity. After about 30 days of induction culture, the resistant adventitious buds were transferred to rooting medium containing 3 mg/L hygromycin B and 200 mg/L Timentin (RIM, 1/2MS basic medium added with 0.05 mg/L IBA and 0.02 mg/L NAA) until adventitious roots were induced. The DNA of rooted plant leaves was extracted, and transgenic poplars were identified by amplifying the sequence of HPT. The Primer3 software was used to design the primer of PagSWEET17a, and the quantitative PCR was carried out by Lightcycler 480 (Roche Applied Science, Penzberg, Upper Bavaria, Germany) to detect the relative expression level of PagSWEET17a in 84k and transgenic poplars. PagActin and PagTubulinin of 84K were selected as internal reference genes to calculate the relative expression of PagSWKET17a. The results showed that the expression levels of transgenic lines OE-1 and OE-2 overexpressing PISWEETI7a were 3000 times and 500 times that of 84K, respectively.

Example 4 Transgenic poplars overexpressing PagSWEET17a promotes wood formation Non-transgenic 84K and transgenic poplars overexpressing PagSWEET17a were grown in greenhouse. Two months old plants were used to collect the data relevant to wood formation. The stem diameter (15cm above the soil) was measured by vernier caliper. The results showed that stem diameter of transgenic poplars overexpressing PagSWEET17a was significantly higher than that of the non-transgenic control (Figure 1). Cross-sections in10th internode of transgenic and control seedlings were made by Leica VT1000 S and observed using Microscope (Olympus). Three plants were detected in each strain, and repeated for 3 times. The results showed that the xylem area of transgenic poplars overexpressing P1SWKET17a was significantly wider than that of the non-transgenic control (Figure 2).

The 16th internode was used for segregation of the fibers in the wood. The bark and pith were removed. The xylem was cut into thin rods with Imm of radial and chordal width and 10 mm of longitudinal length. Select 5-10 rods, put them in a 2 mL centrifuge tube, add 1.8 mL 68502442 the isolated solution (30% hydrogen peroxide: glacial acetic acid: water = 1: 5:4), and put the centrifuge tube in a metal bath at 56°C for 72 h; Wash the isolated samples with distilled water for more than 3 times, stain them with saffron and observe them using ZESS Imager Al microscope, and measure the fiber length and the size of the middle part with Axiovision image processing software. Each sample was measured for 3 times, and 300 fibers were selected for each measurement. The results showed that the fibers of transgenic poplars overexpressing PagSWEET17a were significantly longer than those of non-transgenic control 84k.

Cross-sectional transmission electron microscopy was used to observe the difference of secondary cell wall between transgenic poplars and control. The 15th internode were cut into Smmx5mm cubes, and were placed into a centrifuge tube containing 2.5% glutaraldehyde phosphate buffer (PBS, pH=7.0). The sample were vacuumed to be completely immersed in the solution, and fixed at 4°C for more than 3 hours. Cleaned with 0.1 M PBS buffer for 2-3 times, each time for 10-15 min; Treated with 1% osmic acid solution for 2 h, and wash with phosphate buffer (0.1M, pH7.0) for 2-3 hours. Then, the samples were dehydrated by 30%, 50%, 70%, 80%, 90% and 100% ethanol in turn, and each step was dehydrated for 15 min; Treated with pure acetone twice, 20 min for each time; Treated with the mixture of embedding agent and acetone (v:v=1:1) for 1 h, and then with the mixture of embedding agent and acetone (v : v= 3:1) for 3 h; Finally, pick a single cube to the bottom of a 0.5 ml tube with a toothpick, keep the proper direction, and add pure resin for embedding and high-temperature polymerization. The embedded sample was sliced using the LEICA EM UC7 ultrathin microtome, and 50 nm cross-sections were obtained. The sections were stained by double-staining with uranyl acetate and lead citrate, and 50% ethanol saturated solution for 15min, then observed and photographed by transmission electron microscope (Hitachi H-7650). The results showed that the secondary cell wall of xylem fibers and vessels in transgenic poplars overexpressing PagSWEET17a was significantly thicker than that of the non-transgenic control (Figure 3).

The above-mentioned embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention. On the premise of not departing from the design spirit of the invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the scope of protection determinéd/502442 by the claims of the invention.

Sequence Listing LU502442 <110> Research Institute of Forestry Chinese Academy of Forestry <120> SUGAR TRANSPORTER GENE FOR AFFECTING WOOD FORMATION IN

POPLAR AND APPLICATION THEREOF <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 723 <212> DNA <213> Artificial Sequence <400> 1 atggaaagtc tcagtttgat tatcggagca ctaggtaata ttatttcagt gttactcttt 60 ctttetcctg tgggagtatt tcggcetatt ttgaagcatc gatcgactga agaatttgag 120 agccttectt acatttgcac attgttaaac tettccctet ggacttacta tggtattate 180 aagaccggag agtttctcgt ggccaccata aatgggttte gtattatcet tgagattgtt 240 ttcttgacat tgttccttgt ttttgcacct ccaagaatta gggcaaagac agecatgcett 300 attgggattt tggatgtggg atttctagca gcagcaateg tggtttacca gttactattg 360 cagggtgata tgaagaccga tatcatagga ttcttgggte ctggacttaa cgttgtcatg 420 tatggttctc ctcttgcage catgaaaaca gtggtgagga ctagaagtgt ggagtatatg 480 cctttectte teteectttt cettttettg aatggagggg tetggacttg ctacgecgtg 540 ctaaagaaag attggtttct aggagtggca aatgtggctg gattatttct tggagcagct 600 caaataatac tttatgcaat atattggaaa cccaggtcat caaagaatgc agcatcaaag 660 gattcagagc atgaacacct tctaccatct tccagecatt ttagagaaaa taatgaagca 720 tag LU502442 723

<210> 2 <211> 240 <212> PRT <213> Artificial Sequence <400> 2 Met Glu Ser Leu Ser Leu Ile Ile Gly Ala Leu Gly Asn Ile Ile Ser 1 5 10 15 Val Leu Leu Phe Leu Ser Pro Val Gly Val Phe Arg Arg Ile Leu Lys

His Arg Ser Thr Glu Glu Phe Glu Ser Leu Pro Tyr Ile Cys Thr Leu

40 45 Leu Asn Ser Ser Leu Trp Thr Tyr Tyr Gly Ile Ile Lys Thr Gly Glu 50 55 60 Phe Leu Val Ala Thr Ile Asn Gly Phe Gly Val Val Val Glu Ile Val 65 70 75 80 Phe Leu Thr Leu Phe Leu Val Phe Ala Pro Pro Arg Ile Arg Ala Lys 85 90 95

Thr Ala Met Leu Ile Gly Ile Leu Asp Val Gly Phe Leu Ala Ala Ala

100 105 110 Met Val Val Cys Gln Leu Leu Leu Gln Gly Asp Met Lys Thr Asp Ile

115 120 125 Ile Gly Phe Leu Gly Ala Gly Leu Asn Val Val Met Tyr Gly Ser Pro 130 135 140

Leu Ala Ala Met Lys Thr Val Val Arg Thr Arg Ser Val Glu Tyr Met 145 150 155 160 Pro Phe Leu Leu Ser Leu Phe Val Phe Leu Asn Gly Gly Val Trp Thr

Cys Tyr Ala Val Leu Lys Lys Asp Trp Phe Leu Gly Val Ala Asn Val 180 185 190 Ala Gly Leu Phe Leu Gly Ala Ala Gln Ile Ile Leu Tyr Ala Ile Tyr 195 200 205 Trp Lys Pro Arg Ser Ser Lys Asn Ala Ala Ser Lys Asp Ser Glu His 210 215 220 Glu His Leu Leu Pro Ser Ser Ser His Phe Arg Glu Asn Asn Glu Ala 225 230 235 240 <210> 3 <211> 10820 <212> DNA <213> Artificial Sequence <400> 3 cgtaatcatg gtcatagctg tttcetgtgt gaaattgtta tccgctcaca attccacaca 60 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 120 cattaattgc gttgcgctca ctgcccectt tccagtcggs aaacctgteg tgccagctgc 180 attaatgaat cggccaacgc geggggagag gcggtttecg tattggetag agcagcttgc 240 caacatggtg gagcacgaca ctetcgtcta ctccaagaat atcaaagata cagtctcaga 300 agaccaaagg gctattgaga cttttcaaca aagggtaata tcgggaaacc tectcggatt 360 ccattgccca gctatctetc acttcatcaa aaggacagta gaaaaggaag gtggcaccta 420 caaatgccat cattgcgata aaggaaaggc tatcgttcaa gatgectctg cegacagtgg 480 tcccaaagat ggacceccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 540 gtcttcaaag caagtggatt gatetgataa catggtggag cacgacacte tegtetacte 600 caagaatatc aaagatacag tctcagaaga ccaaagggct attgagactt ttcaacaaag 660 ggtaatatcg ggaaacctec tcggatteca ttgcccagct atctetcact tcatcaaaag 720 gacagtagaa aaggaaggtg gcacctacaa atgccatcat tgcgataaag gaaaggcetat 780 cgttcaagat gectetgeeg acagtggtec caaagatgga cccccaccca cgaggagcat 840 cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgatatetc 900 LU502442 cactgacgta agggatgacg cacaatccca ctatccttcg caagaccttc ctctatataa 960 ggaagttcat ttcatttgga gaggacacgc tgaaatcacc agtctcetcte tacaaatcta 1020 tetetetcga gctttcgcag atcccggggg gcaatgagat atgaaaaagc ctgaactcac 1080 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtetccg acctgatgca 1140 getetcggag ggcgaagaat ctegtgettt cagettegat gtaggagggc gtggatatgt 1200 cctgegggta aatagctgcg cecgatggttt ctacaaagat cgttatgttt atcggcactt 1260 tgcatcggece gegeteecga ttecggaagt gettgacatt ggggagttta gecgagagect 1320 gacctattgc atctccegece gtgeacaggg tgtcacgttg caagacctge ctgaaaccga 1380 actgcccget gttctacaac cggtegegga ggctatggat gegatcgetg cggecgatct 1440 tagccagacg agcggatteg geccattcgg accgcaagga atcggtcaat acactacatg 1500 gcgtgatttc atatgcgcga ttgctgatec ccatgtgtat cactggcaaa ctgtgatgga 1560 cgacaccgtc agtgcetecg tcgegeaggc tetcgatgag ctgatgettt gggecgagga 1620 ctgccccgaa gtecggeace tegtgcacgc ggatttcgge tccaacaatg tectgacgga 1680 caatggccgc ataacagcgg tcattgactg gagcgaggcg atettcgggg atteccaata 1740 cgaggtcgec aacatcttct tetggaggcc gtggttggct tgtatggage agcagacgcg 1800 ctacttcgag cggaggcatc cggagcettge aggatcgeca cgactecggg cgtatatgct 1860 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc 1920 ttgggcgcag ggtcgatgeg acgcaategt ccgatccgga gecgggactg tegggcgtac 1980 acaaatcgcc cgcagaageg cggecgtetg gaccgatgec tgtgtagaag tactcgccga 2040 tagtggaaac cgacgeccca gcactegtec gagggcaaag aaatagagta gatgecgace 2100 ggatctgtcg atcgacaagc tcgagtttct ccataataat gtgtgagtag ttcccagata 2160 agggaattag ggttcctata gggtttcget catgtgttea gcatataaga aacccttagt 2220 atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat 2280 ccagtactaa aatccagatc ccccgaatta attcggegtt aattcagtac attaaaaacg 2340 tccgcaatgt gttattaagt tetctaagcg tcaatttett tacaccacaa tatatcetge 2400 caccagccag ccaacagcte cccgaccgge agetcggeac aaaatcacca ctegatacag 2460 gcagcccatc agtccgggac ggegtcageg ggagagecgt tgtaaggcgg cagactttgc 2520 tcatgttacc gatgctattc ggaagaacgg caactaagct gccggatttg aaacacggat 2580 gatctcgegg agggtagcat gttgattgta acgatgacag agegttgetg cetgtgatca 2640 ccgeggtttc aaaatcggct ccgtegatac tatgttatac gccaactttg aaaacaactt 2700 LU502442 tgaaaaagct gttttctggt atttaaggtt ttagaatgca aggaacagtg aattggagtt 2760 cgtcttgtta taattagctt cttggggtat ctttaaatac tgtagaaaag aggaaggaaa 2820 taataaatgg ctaaaatgag aatatcaccg gaattgaaaa aactgatcga aaaataccgc 2880 tgcgtaaaag atacggaagg aatgtctect getaaggtat ataagetggt gggagaaaat 2940 gaaaacctat atttaaaaat gacggacagc cggtataaag ggaccaccta tgatgtggaa 3000 cgggaaaagg acatgatgct atggctggaa ggaaagctgc ctgttccaaa ggtectgeac 3060 tttgaacggc atgatggctg gagcaatctg ctcatgagtg aggccgatgg cgtectttge 3120 tcggaagagt atgaagatga acaaagccct gaaaagatta tcgagcetgta tgcggagtge 3180 atcaggctct ttcactccat cgacatatcg gattgtcect atacgaatag cttagacagc 3240 cgcttagecg aattggatta cttactgaat aacgatctgg ccgatgtgga ttgcgaaaac 3300 tgggaagaag acactccatt taaagatccg cgcgagctet atgatttttt aaagacggaa 3360 aagcccgaag aggaacttgt cttttcccac ggegacctgg gagacagcaa catctttgtg 3420 aaagatggca aagtaagtgg ctttattgat cttgggagaa gcggcagggc ggacaagtgg 3480 tatgacattg ccttctgegt ccggtegate agggaggata tcggggaaga acagtatgte 3540 gagctatttt ttgacttact ggggatcaag cctgattggg agaaaataaa atattatatt 3600 ttactggatg aattgtttta gtacctagaa tgcatgacca aaatccctta acgtgagttt 3660 tcgttccact gagcetcaga ccccgtagaa aagatcaaag gatcttettg agatectttt 3720 tttetgegeg taatctgctg cttgcaaaca aaaaaaccac cgetaccage ggtggtttgt 3780 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcettcag cagagegeag 3840 ataccaaata ctgtccttct agtgtagecg tagttaggece accacttcaa gaactctgta 3900 gcaccgcceta catacctegc tetgetaate ctgttaccag tggetgetge cagtggegat 3960 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataagge gcagcggteg 4020 ggctgaacgg ggggttcgtg cacacagecc agettggage gaacgaccta caccgaactg 4080 agatacctac agcgtgagct atgagaaagc gecacgcttc ccgaagggag aaaggcggac 4140 aggtatccgg taagcggcag ggtcggaaca ggagagegea cgagggagct tccaggggga 4200 aacgcctggt atctttatag teetgtcggg tttcgecacc tetgacttga gegtegattt 4260 ttgtgatgct cetcaggggg gcggagecta tggaaaaacg ccagcaacge ggcecttttta 4320 cggttectgg ccttttgetg gecttttget cacatgttet ttectgcett atcccctgat 4380 tctgtggata accgtattac cgcectttgag tgagcetgata ccgctcgccg cagecgaacg 4440 accgagceca gcgagtcagt gagcgaggaa gcggaagage gectgatgeg gtattttete 4500 LU502442 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctetcagtac aatctgctet 4560 gatgccgcat agttaagcca gtatacacte cectatcgct acgtgactgg gtcatgectg 4620 cgeeecgaca cecgccaaca cecgetgacg cgcectgacg gecttatetg cteccggcat 4680 ccgcttacag acaagcetgtg accgtetecg ggagctecat gtetcagagg ttttcaccgt 4740 catcaccgaa acgcgcgagg cagggtgect tgatetgggc geecggeggte gagtggegac 4800 ggcgcggctt gtecgcgcec tggtagattg cctggccgta ggecagecat ttttgagegg 4860 ccagcggceg cgataggecg acgegaageg geggggegta gggagegeag cgaccgaagg 4920 gtaggcgctt tttgcagete ttegectatg cgetggecag acagttatge acaggecagg 4980 cgggttttaa gagttttaat aagttttaaa gagttttagg cggaaaaatc gectttttte 5040 tcttttatat cagtcactta catgtgtgac cggttcccaa tgtacggctt tggattecca 5100 atgtacgggt tecgattecc aatgtacgge tttggattec caatgtacgt getatccaca 5160 ggaaagagac cttttcgacc tttttccect gctagggcaa tttgeectag catctgctec 5220 gtacattagg aaccggcgga tgcttcgccc tegatcaget tgcggtageg catgactagg 5280 atcgggccag cetgccccgc ctectectte aaategtact ccggeaggte atttgacccg 5340 atcagcttge gcacgetgaa acagaacttc ttgaactetc cggcectgce actgegtteg 5400 tagatcgtct tgaacaacca tctggcettct gecttgectg cggegeggeg tgecaggcgg 5460 tagagaaaac ggccgatgee gggatcgatc aaaaagtaat cggggtgaac cetcagcacg 5520 tccgggttcet tgecttetgt gatctcgegg tacatccaat cagetagete gatctcgatg 5580 tactccggcc geceggttte gctetttacg atcttgtage gectaatcaa ggcttcacce 5640 tcggataccg tcaccaggeg gccgttettg gecttetteg tacgetgeat ggcaacgtgc 5700 gtggtattta accgaatgca ggtttctace aggtcgtctt tetgetttee gecateggct 5760 cgcecggeaga acttgagtac gtccgeaacg tgtggacgga acacgeggce gggcttatet 5820 cectteectt cccgstateg gttcatggat teggttagat gggaaaccge catcagtace 5880 aggtcgtaat cccacacact ggecatgecg geeggecctg cggaaacctce tacgtgcccg 5940 tctggaagct cgtageggat cacctegcca getegteggt cacgettcga cagacggaaa 6000 acggccacgt ccatgatgct gcgactateg cgggtgecca cetcatagag catcggaacg 6060 aaaaaatctg gttgctcgte geecttggge ggcttectaa tcgacggege accggctgec 6120 ggcggttgee gggattettt gcggattega tcageggecg cttgccacga ttcaccgggg 6180 cgtgcttctg cctecgatgeg ttgeegetgg geggectgeg cggecttcaa cttetccacc 6240 aggtcatcac ccagcgccgc gccgatttet accgggecgg atggtttgcg accgtcacge 6300 LU502442 cgattecteg gecttggggg ttccagtgece attgcagggc cggcagacaa cccagecgct 6360 tacgcctggc caaccgcccg ttectccaca catggggcat tccacggcet cggtgectgg 6420 ttattettga ttttccatge cgectecttt agccgctaaa attcatctac tcatttatte 6480 atttgctcat ttactctggt agctgcgcga tgtattcaga tagcagcteg gtaatggtet 6540 teccttggcg taccgcgtac atettcagct tggtgtgate ctecgccgec aactgaaagt 6600 tgacccgctt catgectggc gtgtectgcca ggctggccaa cattgcagec ttgetgetge 6660 gtecgctegg acggccggca cttagegtgt ttetectttt gctcatttte tetttaccte 6720 attaactcaa atgagttttg atttaatttc agcggecage gectggacct cgcgggcage 6780 gtegcecteg ggttetgatt caagaacggt tgtgccggcg gcggcagtec ctgggtaget 6840 cacgcgctec gtgatacggg actcaagaat gggcagctcg tacccggcca gcgecteggc 6900 aacctcaccg ccgatgegeg tgectttgat cgcecgcgac acgacaaagg cegettgtag 6960 ccttccatee gtgacctcaa tgegetgcett aaccagctec accaggtegg cggtggecca 7020 tatgtcgtaa gggcttggct gcaccggaat cagcacgaag tcggctgect tgatcgegga 7080 cacagccaag tecgccgcct ggggegetee gtegatcact acgaagtcge geeggecgat 7140 ggccttcacg tcgeggteaa tcgtcgggcg gtegatgecg acaacggtta geggttgate 7200 ttcccgcacg gecgeccaat cgegggeact gccctgggga tcggaatcga ctaacagaac 7260 atcggccccg gegagttgea gggcgcggec tagatggett gegatggteg tettgcctga 7320 ccegecttte tggttaagta cagegataac cttcatgegt tecccttgcg tatttgttta 7380 tttactcatc gcatcatata cgcagegacc gcatgacgca agcetgtttta ctcaaataca 7440 catcaccttt ttagacggcg gcgctegett tettcagegg ccaagetgge cggccaggec 7500 gccagcttgg catcagacaa accggccagg atttcatgca gecgeacggt tgagacgtge 7560 gcgggcggct cgaacacgta cccggecgeg atcatetecg cctegatetc ttcggtaatg 7620 aaaaacggtt cetectggce gtectgatgc ggtttcatge ttettectet tggegtteat 7680 tctcggegge cgecagggeg teggectegg tcaatgcete ctcacggaag gcaccgcgcc 7740 gectggcetc ggtgggcgic acttectcgc tgegetcaag tgcgcggtac agggtegage 7800 gatgcacgce aagcagtgca geegectctt tcacggtecg gecttectgg tegatcagct 7860 cecggecetg cgegatctgt gccggestga gegtagggcg ggggccaaac ttcacgecte 7920 gggccttgge ggcetegcec ccgctecggs tecggtegat gattagggaa cgctegaact 7980 Cggcaatgcc ggcgaacacg gtcaacacca tgeggeegge cggegtggtg gtetcggcec 8040 acggctetge caggetacge aggeccgege cggectectg gatgegeteg geaatgteca 8100 LU502442 gtaggtcgcg ggtgctgcgg gecaggeggt ctagectggt cactgtcaca acgtegccag 8160 ggcgtaggtg gtcaagcatc ctggccagct cecgggeggte gegeetggtg ceggtgatcet 8220 tctcggaaaa cagcttggtg cagecggecg cgtgeagttc gecccattgg ttggtcaagt 8280 cctggtegte ggtgctgacg cgggeatage ccageaggece ageggeggcg ctettgttca 8340 tggcgtaatg tetceggtte tagtcgcaag tattctactt tatgecgacta aaacacgega 8400 caagaaaacg ccaggaaaag ggcagggcgg cagectgteg cgtaacttag gacttgtgeg 8460 acatgtcgtt ttcagaagac ggctgcactg aacgtcagaa gecgactgea ctatagcagc 8520 ggaggggttg gatcaaagta ctttgatcce gaggggaacc ctetgattgg catgcacata 8580 caaatggacg aacggataaa ccttttcacg cccttttaaa tatccgttat tctaataaac 8640 gctettttet cttaggttta cccgecaata tatcctgtca aacactgata gtttaaactg 8700 aaggcgggaa acgacaatct gatccaagcet caagctecte tagcattegc cattcagget 8760 gcgcaactet tgggaaggge gatcggtgeg ggectetteg ctattacgee agetggegaa 8820 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttcee agtcacgacg 8880 ttgtaaaacg acggccagtg ccaagcttgg cgtgcctgea ggtcaacatg gtggagcacg 8940 acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa agggcaattg 9000 agacttttca acaaagggta atatccggaa acctcctcgg attccattge ccagctatet 9060 gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc catcattgeg 9120 ataaaggaaa ggccatcgtt gaagatgect ctgccgacag tggtcccaaa gatggaccec 9180 cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 9240 attgatgtga taacatggtg gagcacgaca cacttgtcta ctccaaaaat atcaaagata 9300 cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata tccggaaacc 9360 tccteggatt ccattgccca getatetgte actttattet gaagatagtg gaaaaggaag 9420 gtggctecta caaatgecat cattgcgata aaggaaaggc catcgttgaa gatgectctg 9480 ccgacagtgg tcccaaagat ggacceccac ccacgaggag catcgtggaa aaagaagacg 9540 ttccaaccac gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg 9600 acgcacaatc ccactatcct tcgcaagacc cttectctat ataaggaagt tcatttcatt 9660 tggagaggac ctcgactcta gaggatceee gggtaccggg CCCCCCCLCH aggcgcgcca 9720 agctatcaaa caagtttgta caaaaaagtt ggaatggaaa gtctcagttt gattatcgga 9780 gcactaggta atattatttc agtgttactc tttctttcte ctgtgggagt atttcggegt 9840 attttgaagc atcgatcgac tgaagaattt gagagccttc cttacatttg cacattgtta 9900 LU502442 aactcttecc tgtggactta ctatggtatt atcaagaccg gagagtttct cgtggecace 9960 ataaatgggt ttgstettet cgttgagatt gttttcttga cattgttect tetttttgca 10020 cctccaagaa ttagggcaaa gacagccatg cttattggga ttttggatgt gggetttcta 10080 gcagcagcaa tggtggttte ccagttacta ttgcagggtg atatgaagac cgatatcata 10140 ggattcttgg gtectggact taacgttgtc atgtatggtt ctectettge agccatgaaa 10200 acagtggtea ggactagaag tgtggagtat atgectttcc ttetctecct tttcatttte 10260 ttgaatggag gggtgtggac ttgctacgce gtgctaaaga aagattggtt tctaggagtg 10320 gcaaatgtgg ctggattatt tettggagca gctcaaataa tactttatge aatatattgg 10380 aaacccaggt catcaaagaa tgcagcatca aaggattcag agcatgaaca ccttctacca 10440 tcttccagec attttagaga aaataatgaa gcatagtacc caactttctt gtacaaagtg 10500 gttcgataat tccttaatta actagttcta gageggecge ccaccgeggt ggagetcgaa 10560 tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctettgccgg 10620 tcttgcgatg attatcatat aatttctett gaattacgtt aagcatgtaa taattaacat 10680 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccge aattatacat 10740 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgegegeggt 10800 gtcatctatg ttactgaatt 10820

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 4 ggggacaact ttgtacaaaa aagttggaat ggaaagtctc agtttgat 48

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence LU502442

<400> 5 ggcggccgca caactttgta caagaaagtt gggtactatg cttcattatt ttctc 55

<210> 6

<211> 4719

<212> DNA

<213> Artificial Sequence

<400> 6 gtctgacget cagggaacga cgegtaccge tagccaggaa gagtttgtag aaacgcaaaa 60 aggccatccg tcaggatggce cttetgcetta gtttgatgee tggcagttta tggegggegt 120 cctgeecgee accctecggg cegttgette acaacgttca aatecgctec cggeggattt 180 gtcctactca ggagagegtt caccgacaaa caacagataa aacgaaaggc ccagtcttee 240 gactgagcct ttcgttttat ttgatgcctg gcagttecct actctcgegt taacgctage 300 atggatgttt tcccagtcac gacgttgtaa aacgacggee agtcttaage tcgggcccca 360 aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg atgagcaatg 420 cttttttata atgccaactt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa 480 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca 540 acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt agtcgaccga 600 cagccttcca aatgttcttc gggtgatget gccaacttag tcgaccgaca gecttccaaa 660 tgttcttcte aaacggaatc gtegtatcca gectactege tattgtectc aatgecgtat 720 taaatcataa aaagaaataa ggaaaagagg tgcgagcetc ttttttgtgt gacaaaataa 780 aaacatctac ctattcatat acgctagtgt catagtcctg aaaatcatct gcatcaagaa 840 caatttcaca actcttatac ttttctctta caagtcgttc ggcttcatct ggattttcag 900 cctctatact tactaaacgt gataaagttt ctgtaatttc tactgtatcg acctgcagac 960 tggctgtgta taagggagec tgacatttat attccccaga acatcaggtt aatggegttt 1020 ttgatgtcat tttcgcggtg gctgagatca gecacttctt ccccgataac ggagaccgge 1080 acactggcca tatcggtggt catcatgege cagctttcat ccccgatatg caccaccggg 1140 LU502442 taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccate 1200 cgtcgcccgg gegtgtcaat aatatcacte tgtacatcca caaacagacg ataacggcetc 1260 tctettttat aggtgtaaac cttaaactgc atttcaccag cecctattet cetcagcaaa 1320 agagccgttc atttcaataa accgggcgac ctcagccate cettectgat tttecgcttt 1380 ccagcgttcg gcacgcagac gacgggctte attctgeatg gttgtgctta ccagaccgga 1440 gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac tgtcactgta 1500 atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata tcagtatata 1560 ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct tttagtaagc 1620 cggatccacg cggcegtttac gcccccccetg ccactcateg cagtactgtt gtaattcatt 1680 aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 1740 catcagcacc ttgtcgectt gegtataata tttgcccatg gtgaaaacgg gggcgaagaa 1800 gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggetga 1860 gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 1920 cgccacatct tgcgaatata tgtgtagaaa ctgecggaaa tecgtegtggt attcacteca 1980 gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactate 2040 ccatatcacc agctcaccgt ctttcattge catacggaat tecggatgag cattcatcag 2100 gcgggcaaga atgtgaataa aggccggata aaacttgtge ttatttttet ttacggtctt 2160 taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2220 aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 2280 gatttttttc tccattttag cttccttage tcctgaaaat ctegataact caaaaaatac 2340 gcccggtagt gatcttattt cattatggte aaagttggaa cctettacgt gccgatcaac 2400 gtctcatttt ceccaaaagt tggcccaggg ctteccggta tcaacaggga caccaggatt 2460 tatttattct gcgaagtgat cttccgtcac aggtatttat teggcgcaaa gtgegtcggg 2520 tgatgctgece aacttagtcg actacaggtc actaatacca tctaagtagt tgattcatag 2580 tgactggata tattetettt tacagtatta tgtagtctgt tttttatgca aaatctaatt 2640 taatatattg atatttatat cattttacgt ttctcgttca getttcttgt acaaagttgg 2700 cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa 2760 taaaatcatt atttgccatc cagctgatat cccctatagt gagtcgtatt acatggtcat 2820 agctgtttce tggcagctet ggeecgtgte tcaaaatcte tgatgttaca ttgcacaaga 2880 taaaaatata tcatcatgtt agaaaaactc atcgagcatc aaatgaaact gcaatttatt 2940 LU502442 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa 3000 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtcetgega ttecgacteg 3060 tccaacatca atacaaccta ttaatttcce ctcgtcaaaa ataaggttat caagtgagaa 3120 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttetttcca 3180 gacttgttca acaggccagc cattacgcte gtcatcaaaa tcactcgeat caaccaaacc 3240 gttattcatt cetgattgcg cctgagegag acgaaatacg cgatcgctgt taaaaggaca 3300 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt 3360 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggategcagt 3420 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat 3480 aaattccgtc agccagttta gtctgaccat ctcatetgta acatcattgg caacgctace 3540 tttgccatgt ttcagaaaca actctggcgc atcgggcettc ccatacaatc gatagattgt 3600 cgcacctgat tgcccgacat tatcgegage ccatttatac ccatataaat cagcatccat 3660 gttggaattt aatcgcggcc tegagcaaga cgtttcecgt tgaatatggc tcatagatcet 3720 tttetccatc actgataggg agtggtaaaa taactccatc aatgatagag tgtcaacaac 3780 atgaccaaaa teccttaacg tgagttacgc gtattaattg cgttgegcete actgeeccget 3840 ttccagtcgg gaaacctgte gteccagctg cattaatgaa tcggecaacg cgeggggaga 3900 ggcggtttge gtattgggcg ctettecgct tectegetea ctgactegct gegeteggte 3960 gttcggetge ggcgageggt atcagetcac tcaaaggegg taatacggtt atccacagaa 4020 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggce caggaaccgt 4080 aaaaaggceg cattectggc gtttttccat aggctecgec cecctgacga geatcacaaa 4140 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggegttt 4200 ccccctggaa gctecctegt gegetetect gttecgacce tgccecttac cggatacctg 4260 tecgcetttc teccttcggg aagegtggceg ctttctcaat gctcacectg taggtatcte 4320 agttcggtat aggtcgttcg ctccaagctg gectetetec acgaaceeec cgttcageee 4380 gaccgcetgeg cettatccgg taactatcet cttgagtecca acccggtaag acacgactta 4440 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggeggtgct 4500 acagagttct tgaagtgetg gectaactac ggttacacta gaagaacagt atttggtatc 4560 tecgctctgc tgaagccagt taccttcgga aaaagagttg gtagcetcttg atccggcaaa 4620 caaaccaccg ctggtagcgg tggttttttt gtttgcaage agcagattac gcgcagaaaa 4680 aaaggatctc aagaagatcc tttgatcttt tctacgggg 4719 LU502442 <210> 7 <211> 11752 <212> DNA <213> Artificial Sequence <400> 7 aattcagtaa catagatgac accgcgcecg ataatttatc ctagtttgcg cectatattt 60 tgttttctat cecstattaa atgtataatt gcgggactct aatcataaaa acccatctca 120 taaataacgt catgcattac atgttaatta ttacatgctt aacgtaattc aacagaaatt 180 atatgataat catcgcaaga ccggcaacag gattcaatct taagaaactt tattgccaaa 240 tgtttgaacg atcggggaaa ttegagctec accgeggteg geggecgcete tagaactagt 300 taattaagga attatcgaac cactttgtac aagaaagctg aacgagaaac gtaaaatgat 360 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa 420 acacaacata tccagtcact atggtcgacc tgecagactgg ctgtgtataa gggagectga 480 catttatatt ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgeggtggct 540 gagatcagcc acttettecc cgataacgga gaccggcaca ctggecatat cggtggtcat 600 catgcgccag ctttcatcce cgatatgcac caccgggtaa agttcacggg agactttatc 660 tgacagcaga cgtgcactgg ccagggggat caccatccgt cgcccggecg tgtcaataat 720 atcactctgt acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt 780 aaactgcatt tcaccagtcc ctgttctcgt cagcaaaaga gccgttcatt tcaataaacc 840 gggcgacctc agecatecct tectgatttt ccgctttcca gegttcggea cgcagacgac 900 gggcttcatt ctgcatgett gtgcttacca gaccggagat attgacatca tatatgectt 960 gagcaactga tagctetcgc tgtcaactgt cactgtaata cgetgettca tagcacacct 1020 ctttttgaca tacttcgggt atacatatca gtatatattc ttataccgca aaaatcagcg 1080 cgcaaatacg catactgtta tctggctttt agtaagccgg atcctctaga ttacgccccg 1140 cectgccact catcgcagta ctgttgtaat tcattaagca ttctgecgac atggaageca 1200 tcacagacgg catgatgaac ctgaatcgcc agcggcatca geaccttgte gecttgegta 1260 taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa 1320 tcaaaactgg tgaaactcac ccagegattg gctgagacga aaaacatatt ctcaataaac 1380 LU502442 cctttaggga aataggccag gttttcaccg taacacgcca catettgcga atatatgtgt 1440 agaaactgcc ggaaatcgte gtggtattca ctccagagcg atgaaaacgt ttcagtttgc 1500 tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagcte accgtettte 1560 attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggec 1620 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccegtaat atccagctga 1680 acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga 1740 tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagettec 1800 ttagctectg aaaatctcge cggatectaa ctcaaaatce acacattata cgagecggaa 1860 gcataaagtg taaagcctgg ggtgcctaat gcggccgcca tagtgactgg atatettetg 1920 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta 1980 tatcatttta cgtttctcgt tcagcttttt tgtacaaact tgtttgatag cttggcgcgc 2040 ctcgaggggg ggcccggtac ccggggatcc tctagagteg aggtectete caaatgaaat 2100 gaacttcctt atatagagga agggtcttge gaaggatagt gggattgtec gtcatecctt 2160 acgtcagtgg agatatcaca tcaatccact tectttgaag acgtggttgg aacgtcttct 2220 ttttccacga tectectcet gggtgggggt ccatetttgg gaccactgte ggcagaggca 2280 tcttcaacga tggcetttec tttatcgcaa tgatggcatt tgtaggagec accttecttt 2340 tccactatct tcacaataaa gtgacagata getgggcaat ggaatecgag gaggtttecg 2400 gatattaccc tttgttgaaa agtctcaatt gccctttget cttctgagac tgtatctttg 2460 atatttttgg agtagacaag tgtgtcgtgc tccaccatgt tatcacatca atccacttge 2520 tttgaagacg tggttggaac gtcttctttt tccacgatge tectegtggg tggggetcca 2580 tetttgggac cactetcggc agaggcatct tcaacgatgg cctttecttt atcgcaatga 2640 tggcatttgt aggagccacc ttecttttec actatettca caataaagtg acagatagct 2700 gggcaatgga atccgaggag gtttccggat attacccttt gttgaaaagt ctcaattgcc 2760 ctttggtctt ctgagactgt atctttgata tttttggagt agacaagtgt gtegtgctec 2820 accatgttga cctgcaggcea cgccaagctt ggcactggcc gtegttttac aacgtegtga 2880 ctgggaaaac cctggegtta cccaacttaa tegccttgca gcacatecec ctttegecag 2940 ctggcgtaat agcgaagagg cecgcaccga tcgeecttee caacagttge gcagcctgaa 3000 tggcgaatge tagagcagct tgagettgga tcagattgte gtttcccgce ttcagtttaa 3060 actatcagtg tttgacagga tatattggcg ggtaaaccta agagaaaaga gegtttatta 3120 gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tetatetgca 3180 LU502442 tgccaaccac agggtteccec tcgggatcaa agtactttga tccaaccect ccgctgctat 3240 agtgcagtcg gcttetgacg ttcagtgeag cegtettctg aaaacgacat gtcgcacaag 3300 tcctaagtta cgcgacaggc tgecgecectg cecttttect ggegttttet tgtegegtgt 3360 tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacge catgaacaag 3420 agcgccgceg ctggcctect gggetatgee cgegtcagea ccgacgacca ggacttgacc 3480 aaccaacggg cegaactgca cgeggeeggc tgcaccaagc tgttttccga gaagatcacc 3540 ggcaccaggc gegaccgece ggagctggce aggatecttg accacctacg cectggcgac 3600 gttetgacag tgaccagegct agaccgectg geccgcagca cecgcgacct actggacatt 3660 gccgagcgca tccaggaggc cggcgcggec ctgcgtagec tggcagagee gtgggecgac 3720 accaccacgc cggccgeceg catggtattg accgtgttcg ccggeattge cgagttcgag 3780 cgttcectaa tcatcgaccg cacccggagc gggcgcgagg ccgecaagge ccgaggegte 3840 aagtttggcc cecgcectac cctcaccecg gcacagateg cgeacgeccg cgagcetgate 3900 gaccaggaag gccgcaccgt gaaagaggcg getgeactge ttggcgtgca tegetcgace 3960 ctgtaccgcg cacttgageg cagcgaggaa gtgacgecca ccgaggecag geggegeggt 4020 gcctteegtg aggacgcatt gaccgaggce gacgeectgg cggecgecga gaatgaacgc 4080 caagaggaac aagcatgaaa ccgecaccagg acggccagga cgaaccgttt ttcattaccg 4140 aagagatcga ggcggagatg atcgcggceg ggtacgtgtt cgagecgcec gegeacgtcet 4200 caaccgtgcg getgeatgaa atcctggecg gtttgtetga tgccaagctg geggectgge 4260 cggccagctt ggccgctgaa gaaaccgage gecgecgtct aaaaaggtga tgtgtatttg 4320 agtaaaacag cttgcgtcat gcggtcgetg cgtatatgat gegatgagta aataaacaaa 4380 tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa 4440 gacgaccatc gcaacccate tageccgcec cctgcaacte geeggggecg atgttetgtt 4500 agtcgattcc gatccccagg gcagtgcccg cgattgggcg geegtgeggg aagatcaacc 4560 gctaaccgtt gtcggcatcg accgcecgac gattgaccgc gacgtgaagg ccatcggecg 4620 gcgcgacttc gtagtgatcg acggagegee ccaggeggceg gacttggetg tetecgcgat 4680 caaggcagce gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac 4740 cgccgacctg gtggagcetgg ttaagcageg cattgaggtc acggatggaa ggctacaage 4800 ggcctttgte gtgtcgeggge cgatcaaagg cacgegeate ggcggtgagg ttgccgaggc 4860 gctggccggg tacgagetge ccattettga gteccgtate acgcagcgcg tgagetaccee 4920 aggcactgce gccgccggca caaccgttet tgaatcagaa cccgagggcg acgetgeccg 4980 LU502442 cgaggtccag gegetggecg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa 5040 gagaaaatga gcaaaagcac aaacacgcta agtgecggec gtecgagcgc acgcagcage 5100 aaggctgcaa cattggccag cetggcagac acgccagcca tgaagegggt caactttcag 5160 ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt 5220 accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat 5280 gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac 5340 cgacgcegtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taageggctg 5400 gettgtectgc cggcectgca atggeactgg aacccccaag cccgaggaat cggegtgacg 5460 gtcgcaaacc atccggcccg gtacaaatcg gegeggegct gggtgatgac ctggtggaga 5520 agttgaaggc cgcgcaggec geeccagegge aacgcatega ggcagaagca cgceccggtg 5580 aatcgtggca agcggccect gatcgaatce gcaaagaatc ccggeaaccg ccggeagecg 5640 gtecgcegte gattaggaag ccgeccaagg gegacgagea accagatttt ttegttecga 5700 tectctatga cgtgggcace cgegatagte gcagcatcat ggacgtggece gttttecgte 5760 tgtcgaageg tgaccgacga getggegagg tgatcegeta cgagcttcca gacgggcacg 5820 tagaggtttc cgcagggecg gccggcatgg ccagtatetg ggattacgac ctggtactga 5880 tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaage 5940 ccggeegegt gttecgteca cacgttgegg acgtactcaa gttetgeegg cgagecgatg 6000 gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgeacgttg 6060 ccatgcagcg tacgaagaag gccaagaacg geecgectggt gacggtatce gagggtgaag 6120 ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg geggecggag tacatcgaga 6180 tcgagctage tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga 6240 cggttcaccce cgattacttt ttgatcgatc ccggeatcgg cegttttete taccgectgg 6300 cacgccgcgc cgeaggeaag gcagaagcca gatggttatt caagacgate tacgaacgca 6360 gtggcagcgc cggagagttc aagaagttct gtttcaccgt gecgecaagcetg atcgggtcaa 6420 atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggetggeecg atectagtca 6480 tecectaccg caacctgatc gagggcgaag catecgccgg ttectaatgt acggagcaga 6540 tectagggca aattgeccta gcaggggaaa aaggtcgaaa aggtctettt cctgtggata 6600 gcacgtacat tgggaaccca aagecgtaca ttgggaaccg gaacccgtac attgggaacc 6660 caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag 6720 gcgatttttc cgectaaaac tctttaaaac ttattaaaac tcttaaaacc cgectggect 6780 LU502442 gtecataact gtctggccag cgcacagceg aagagctgca aaaagegect accctteggt 6840 cectgcgcte cctacgeece gccgcttcgc gteggectat cgeggecget ggccgctcaa 6900 aaatggctgg cctacggcca ggeaatctac cagggegegg acaagecgeg cegtegeeac 6960 tegaccgceg gcgcccacat caaggeacce tgectcgege gtttcggtea tgacggtgaa 7020 aacctctgac acatgcagct cccggagacg gtcacagctt gtetgtaage ggatgccggg 7080 agcagacaag cccgtcaggg cgegtcageg ggtettggcg ggtatcgggg cgcagccatg 7140 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 7200 ttgtactgag agtgcaccat atgcggtetg aaataccgca cagatgegta aggagaaaat 7260 accgcatcag gcgctettec gettectege tcactgacte getgegeteg gtegttcgge 7320 tgcggegage ggtatcaget cactcaaagg cggtaatacg gttatccaca gaatcagggg 7380 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 7440 ccgegttgct ggcatttttc cataggctec geccccctga cgagcatcac aaaaatcgac 7500 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 7560 gaagctecct cgtgegetcet cetgttecga cectgccect taccggatac ctgtecgect 7620 ttcteecttc gegaagcetg gegetttete atagctcacg ctgtaggtat ctcagttcgg 7680 tgtaggtcgt tcgctccaag ctggectatg tgcacgaace ceccattcag cecgaccgct 7740 gcgccttatc cggtaactat cgtettgagt ccaacccggt aagacacgac ttatcgecac 7800 tggcagcage cactggtaac aggattagca gagcgaggta tgtaggeggt gctacagagt 7860 tettgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgegete 7920 tectgaagcc agttaccttc ggaaaaagag ttggtagetce ttgatecggc aaacaaacca 7980 ccgctggtag cggtggtttt tttetttgca agcagcagat tacgcgcaga aaaaaaggat 8040 ctcaagaaga tcctttgatc ttttctacgg gatectgacgc tcagtggaac gaaaactcac 8100 gttaagggat tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat 8160 attttatttt ctcccaatca ggcttgatec ccagtaagtc aaaaaatagce tegacatact 8220 gttetteccc gatatectec ctgatcgace ggacgcagaa ggcaatgtca taccacttgt 8280 ccgceccetgec gcttetecca agatcaataa agccacttac tttgecatct ttcacaaaga 8340 tgttgctgte teccagetcg cegtgggaaa agacaagtte ctettcggec ttttccatet 8400 ttaaaaaatc atacagctcg cecggatett taaatggagt gtettettec cagttttcge 8460 aatccacatc ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctetcta 8520 agctattcgt atagggacaa tccgatatgt cgatggagtg aaagagectg atgcactecg 8580 LU502442 catacagctc gataatcttt tcagggcttt gttcatcttc atactettec gagcaaagga 8640 cgccatcggce ctcactcatg agcagattge tccagecate atgecgttca aagtgcagga 8700 cctttggaac aggcagcttt ccttccagece atagcatcat gtecttttce cgttccacat 8760 cataggtggt ccctttatac cggetgtecg tcatttttaa atataggttt tcattttetc 8820 ccaccagctt atatacctta gcaggagaca ttccttecgt atcttttacg cageggtatt 8880 tttcgatcag ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc 8940 ctettttcta cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa 9000 ttcactgttc cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt 9060 ttcaaagttg gcgtataaca tagtatcgac ggagecgatt ttgaaaccgc ggtgatcaca 9120 ggcagcaacg ctetgteate gttacaatca acatgctacc ctccgegaga tcatecgtet 9180 ttcaaacccg gcagcttagt tgccattett ccgaatagea tcggtaacat gagcaaagtc 9240 tgeegectta caacggcetcet cccgctgacg cegteccgga ctgatgggct gectgtateg 9300 agtggtgatt ttgtgccgag ctgccggteg gggagetgtt gectggcteg tggcaggata 9360 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgeg gacgttttta 9420 atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt 9480 tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg 9540 tttcttatat gctcaacaca tgagcgaaac cctataggaa cectaattec cttatctggg 9600 aactactcac acattattat ggagaaactc gagcttgtcg atcgacagat ccggteggea 9660 tctactctat ttctttgeee tcggacgagt getggggecgt cggtttecac tatcggegag 9720 tacttctaca cagccatcgg tccagacgge cgegcttetg cgggcgattt gtgtacgecee 9780 gacagteccg getecggate ggacgattge gtegeatcga cectgcgcec aagetgeate 9840 atcgaaattg ccgtcaacca agctetgata gagttggtca agaccaatge ggagcatata 9900 cgcccggagt cgtggegate ctgecaagcete cggatgecte cgetcgaagt agegegtetg 9960 ctgctccata caagccaacc acggectcca gaagaagatg ttggegacct cgtattggga 10020 atccccgaac atcgectege tccagtcaat gaccectett atgeggecat tetecgtcag 10080 gacattgttg gagccgaaat ccgegtgeac gaggtgecgg acttcggggc agtectegge 10140 ccaaagcatc agctcatcga gagectgege gacggacgca ctgacggtgt cgtecatcac 10200 agtttgccag tgatacacat ggggatcagce aatcgcgcat atgaaatcac gccatgtagt 10260 gtattgaccg attccttgeg gtecgaatgg gecgaacceceg ctegtetgge taagatcgge 10320 cgcagcgatc gcatccatag cctccgegac cggttgtaga acagegggca gtteggttte 10380 LU502442 aggcaggtct tgcaacgtga caccctgtge acggegggag atgcaatagg tcaggctete 10440 gctaaactec ccaatgtcaa geacttccgg aatcgggage geggecgatg caaagtgecg 10500 ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatec 10560 acgccectect acatcgaage tgaaagcacg agattcttcg cectecgaga gctgcatcag 10620 gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagtte 10680 aggctttttc atatctcatt gecccccggg atctgegaaa getcgagaga gatagatttg 10740 tagagagaga ctggtgattt cagcgtgtce tetccaaatg aaatgaactt ccttatatag 10800 aggaaggtct tgcgaaggat agtgggattg tgegtcatce cttacgtcag tggagatate 10860 acatcaatcc acttgctttg aagacgtggt tggaacgtct tetttttcca cgatgctect 10920 catggetggg gotccatctt tgggaccact gtcggcagag gcatettgaa cgatagectt 10980 tcctttatcg caatgatggc atttgtaggt gecaccttec ttttctactg tecttttgat 11040 gaagtgacag atagctgggc aatggaatec gaggaggttt cccgatatta cectttgttg 11100 aaaagtctca atageccttt ggtcttctga gactgtatct ttgatattct tggagtagac 11160 gagagtgtcg tgctccacca tgttatcaca tcaatccact tectttgaag acgtggtteg 11220 aacgtcttct ttttccacga tgetectegt gggtgggggt ccatctttgg gaccactgte 11280 ggcagaggca tcttgaacga tagectttee tttatcgcaa tgatggcatt tgtaggtgec 11340 accttccttt tctactgtee ttttgatgaa gtgacagata gctgggcaat ggaatecgag 11400 gaggtttecc gatattacce tttgttgaaa agtctcaata geectttggt cttetgagac 11460 tgtatctttg atattcttgg agtagacgag agtgtcgtec tccaccatgt tggcaagctg 11520 ctctagccaa tacgcaaacc gccteteccec gegegttgge cgattcatta atgcagctgg 11580 cacgacaggt ttcccgactg gaaagcgggc agtgagegea acgcaattaa tgtgagttag 11640 ctcactcatt aggcacccca ggctttacac tttatgettc cggetcgtat ettetetgga 11700 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cg 11752

Claims (10)

1. A sugar transporter gene for affecting wood formation in poplar, characterized in that a nucleotide sequence is shown in SEQ ID NO: 1.

2. A protein encoded by the sugar transporter gene according to claim 1, characterized in that an amino acid sequence is shown in SEQ ID NO: 2.

3. A pair of primers for amplifying the sugar transporter gene according to claim 1, characterized in that the primers comprises an upstream primer in SEQ ID NO: 4 and a downstream primer in SEQ ID NO: 5.

4. A recombinant vector, characterized by comprising the sugar transporter gene of claim 1.

5. The recombinant vector according to claim 4, characterized in that a nucleotide sequence is shown in SEQ ID NO: 3.

6. A recombinant bacterium, characterized by comprising the recombinant vector of claim 4 or 5.

7. A method for constructing the recombinant vector according to claim 4 or 5, characterized by comprising the following steps: (1) PCR amplification by the pair of primers of claim 3 with poplar cDNA as a template to obtain the sugar transporter gene of claim 1; (2) Carrying out a recombination reaction on the sugar transporter gene and an entry vector to obtain a recombinant entry vector, (3) Performing LR reaction to introduce the sugar transporter gene into the plant expression vector, assembling the constitutive promoter p35S at the 5' end of the sugar transporter gene and the strong terminator NOS at the 3' end of the sugar transporter gene to obtain the recombinant vector.

8. An application of the sugar transporter gene according to claim 1, protein according to claim 2, primers according to claim 3, recombinant vector according to any one of claims 4 and 5 or recombinant bacteria according to claim 6 in affecting the growth and development of poplar.

9. The application according to claim 8, characterized in that the application is specifically an application in affecting wood formation in poplar.

10. The application according to claim 9, characterized in that the affecting wood formatid1)502442 includes promoting the widening of xylem and thickening of secondary cell wall in xylem fibrocyte.