本發(fā)明屬于分子生物學(xué)領(lǐng)域，具體涉及鹽膚木rch2hd基因。

背景技術(shù)：

五倍子是一種重要的化工原料，是由半翅目hemiptera，蚜總科aphidoidea,癭綿科pemphigidae，倍蚜族melaphidini的五倍子蚜蟲生活在漆樹科anacardiaceae鹽膚木屬rhus的寄主植物上，刺激葉片組織增生而形成的蟲癭。中國五倍子利用有2000多年的歷史，古代主要用于中藥，現(xiàn)代廣泛用于化工、醫(yī)藥、紡織、食品等行業(yè)，具有重要的經(jīng)濟(jì)價(jià)值。鹽膚木rhuschinensis是角倍蚜schlechtendaliachinensis的主要寄主，角倍是角倍蚜在寄生在鹽膚木上形成的蟲癭，是我國主要的五倍子種類之一，其產(chǎn)量占我國五倍子總產(chǎn)量的70%以上。

角倍的主要活性成分是單寧，單寧（tannins）又稱單寧酸、鞣質(zhì)，是植物體內(nèi)一大類多酚黃酮化合物，單寧的生物合成途徑已基本明確,主要是通過莽草酸途徑合成,即由莽草酸途徑形成苯丙氨酸，再經(jīng)一系列反應(yīng)后最終形成單寧。許多植物中黃酮生物合成途徑中關(guān)鍵酶基因的生化功能已經(jīng)得到驗(yàn)證，關(guān)鍵酶包括黃酮3-o-單氧酶、花青素3-o-葡糖基轉(zhuǎn)移酶、無花色素還原酶（lar），花色素還原酶（anr），二氫黃酮醇4-還原酶（dfr）、查兒酮合成酶（chs）和查兒酮異構(gòu)酶（chi）等。黃酮3-o-單氧酶在角倍的單寧合成過程中起到至關(guān)重要的調(diào)節(jié)作用，所以，鹽膚木黃酮3-o-單氧酶的表達(dá)水平對(duì)其產(chǎn)生角倍的單寧含量起著重要的調(diào)節(jié)作用，黃酮3-o-單氧酶基因是控制植物黃酮3-o-單氧酶表達(dá)的遺傳基礎(chǔ)。只有找到鹽膚木黃酮3-o-單氧酶的基因，才能對(duì)鹽膚木進(jìn)行遺傳改良，從而提高角倍中單寧含量。

技術(shù)實(shí)現(xiàn)要素：

為解決上述問題，本發(fā)明通過對(duì)鹽膚木進(jìn)行接種，并定期收集不同發(fā)育階段的蟲癭分別提取rna，通過轉(zhuǎn)錄組測序分析、基因注釋、kegg數(shù)據(jù)庫比對(duì)等步驟，得到了鹽膚木單寧合成關(guān)鍵基因rch2hd（序列1、2），并采用qpcr技術(shù)對(duì)rch2hd基因進(jìn)行驗(yàn)證（表2，圖4），具體技術(shù)方案如下：

1、樣本收集與處理

將五倍子蚜蟲放于鹽膚木上（rhuschinensismill），五倍子蚜蟲刺激葉翅產(chǎn)生角倍，定期收集生長于鹽膚木葉翅上不同生長時(shí)期的角倍，每次收集的角倍清除角倍內(nèi)所有蚜蟲后分為兩部分保存，一部分冷凍保存，用于提取總rna，另一部分用于測定單寧含量。

2、測序及轉(zhuǎn)錄組分析

提取去除蚜蟲后的五倍子的總rna，檢測rna質(zhì)量及純度后進(jìn)行rna轉(zhuǎn)錄組測序，序列經(jīng)閱讀質(zhì)量分析、統(tǒng)計(jì)學(xué)比對(duì)分析、測序飽和度分析、參考基因讀取分布及參考基因組讀取分布分析后得到高質(zhì)量鹽膚木轉(zhuǎn)錄組序列。

3、基因注釋與篩選

拼接得到的所有功能基因都分別注釋到ncbi數(shù)據(jù)庫中的nr數(shù)據(jù)庫、nt數(shù)據(jù)庫及swiss-prot數(shù)據(jù)庫、kegg數(shù)據(jù)庫、cog數(shù)據(jù)庫、go數(shù)據(jù)庫中，得到與鹽膚木單寧合成通路相關(guān)基因，并結(jié)合基因表達(dá)量與單寧含量同步一致性，最終篩選出鹽膚木單寧合成的關(guān)鍵基因rch2hd（dna序列及其對(duì)應(yīng)的蛋白質(zhì)序列如序列3所示）。

4、基因表達(dá)量驗(yàn)證

為了驗(yàn)證rch2hd基因，將rch2hd基因做實(shí)時(shí)熒光定量（qpcr）分析，采用相對(duì)定量方法分析rch2hd基因相對(duì)表達(dá)量(圖4)。

附圖說明

圖1為鹽膚木rhuschinensis5個(gè)不同發(fā)育時(shí)期單寧含量；

圖2為鹽膚木rch2hd基因qpcr相對(duì)定量結(jié)果；

圖3為鹽膚木rch2hd基因qpcr相對(duì)表達(dá)量驗(yàn)證結(jié)果；圖中縱坐標(biāo)為rch2hd基因qpcr相對(duì)表達(dá)量，橫坐標(biāo)為鹽膚木發(fā)育的5個(gè)不同時(shí)間點(diǎn)（從6月21-8月28日）；

圖4為鹽膚木不同發(fā)育時(shí)期單寧含量動(dòng)態(tài)變化圖；圖中縱坐標(biāo)為單寧含量（單寧占鹽膚木倍子重量的百分?jǐn)?shù)），橫坐標(biāo)為鹽膚木發(fā)育的5個(gè)不同時(shí)間點(diǎn)（從6月21-8月28日），5個(gè)時(shí)間點(diǎn)鹽膚木分別進(jìn)行rna轉(zhuǎn)錄組測序分析。

具體實(shí)施方式

1、樣本收集

五倍子蚜蟲飼養(yǎng)于中國西南生態(tài)研究中心資源昆蟲研究所中的鹽膚木上（rhuschinensismill）。每隔十五天收集一次生長于鹽膚木葉翅上不同生長時(shí)期的角倍，共計(jì)8次。每次收集的角倍清除角倍內(nèi)所有蚜蟲后分為兩部分保存，一部分先以液氮凍存，隨后移入?80°c冰箱中以便提取總rna，另一部分用于單寧含量的測定。

2、測序及轉(zhuǎn)錄組分析

去除蚜蟲后的五倍子以通用植物總rna提取試劑盒（離心柱型）（百泰克，中國）提取總rna。以1%的瓊脂糖凝膠電泳檢測所提總rna質(zhì)量。以nanophotometer?分光光度計(jì)(implen,ca,usa)檢測純度，以核糖核酸6000納米盒(agilenttechnologies,ca,usa)檢測rna完整度。以打斷后的mrna為模板，隨機(jī)六聚體引物合成一鏈cdna，然后配制雙鏈合成反應(yīng)體系合成雙鏈cdna。合成的雙鏈cdna以qiaquickpcrextractionkit純化，經(jīng)過磁珠純化、粘性末端修復(fù)、3’末端加堿基a、加測序接頭后，進(jìn)行pcr擴(kuò)增，從而完成整個(gè)文庫制備工作。

去除含接頭的片段，其余所需片段以瓊脂糖凝膠電泳純化并進(jìn)行pcr擴(kuò)增。每個(gè)擴(kuò)增樣品以nebnext?ultra?directionalrnalibraryprep試劑盒進(jìn)行測序(neb,usa)，所得基因代碼整合為測序文庫。整合后，文庫以illuminahiseqtm2000測序平臺(tái)雙向測序。最后，構(gòu)建好的文庫以ampurexp體系純化并以安捷倫生物分析儀2100系統(tǒng)(agilenttechnologies,ca,usa)檢測。經(jīng)測序儀讀取后，將去除含n（表示無法確定堿基信息）比例大于10%及低質(zhì)量的片段(質(zhì)量值q≤5的堿基數(shù)占整條read的50％以上)。

轉(zhuǎn)錄組序列拼接時(shí)將trinity2軟件中的min_kmer_cov選項(xiàng)設(shè)置為2，其余所有參數(shù)均為默認(rèn)值。序列經(jīng)閱讀質(zhì)量分析、統(tǒng)計(jì)學(xué)比對(duì)分析、測序飽和度分析、參考基因讀取分布及參考基因組讀取分布分析后得到。

3、基因注釋

拼接得到的所有功能基因都分別注釋到ncbi數(shù)據(jù)庫中的nr數(shù)據(jù)庫、nt數(shù)據(jù)庫及swiss-prot數(shù)據(jù)庫、kegg數(shù)據(jù)庫、cog數(shù)據(jù)庫、go數(shù)據(jù)庫中?；虮磉_(dá)水平為每條基因堿基讀取比對(duì)到參考基因圖譜上的堿基數(shù)量，以rpkm表示1，rpkm值的大小反映了基因表達(dá)的豐度。將rpkm>0.3設(shè)為基因顯著表達(dá)閾值。fpkm法能消除基因長度和測序量差異對(duì)計(jì)算基因表達(dá)量的影響，計(jì)算得到的基因表達(dá)量可直接用于比較不同樣品間的基因差異表達(dá)。

4、實(shí)時(shí)熒光定量分析

為了驗(yàn)證rna分析結(jié)果，實(shí)時(shí)熒光定量采用icycler熒光實(shí)時(shí)檢測系統(tǒng)(bio-rad,hercules,ca)，采用相對(duì)定量方法分析各目的基因相對(duì)表達(dá)量。反應(yīng)體系均為25ul，其中，每個(gè)反應(yīng)體系中包含以1/40(v/v)稀釋過的的模板cdna2ul，及終濃度各為0.5mm的上下游引物。實(shí)時(shí)熒光定量反應(yīng)條件為：95°c，5分鐘；95°c，20秒；62°c，45秒；45個(gè)循環(huán)。最后，以熔解曲線檢測擴(kuò)增特異性，熔解曲線反應(yīng)條件為：變性后樣品冷卻至55°c，然后以每10秒升高0.5°c為1個(gè)循環(huán)，重復(fù)80個(gè)循環(huán)，直至95°c。pcr產(chǎn)物以1.5%瓊脂糖凝膠電泳檢測，隨后純化擴(kuò)增產(chǎn)物并測序，驗(yàn)證有效擴(kuò)增。以actin為內(nèi)參基因校準(zhǔn)模板濃度。每個(gè)處理重復(fù)三次。

序列1.鹽膚木rch2hd基因序列（2094bp）

aaacaatttttagggcccttggccctacttcatcattgatacaaagaactcaacctttag60

tccacaacaatatatcagctttctcccaagcttaaaacgaaagtaacttgtgacatttct120

caagaaatttaaaagtcttttaggagacagaataaccacttaaactatttatgtccatag180

tttatgatattacaagcaacatttattggagatacccatgaagcataatttttctacaaa240

gtcaaaaagataacatgaccaagagcctgttttctgtttcgtcatcatcagagaatcata300

cttacagcttactcctctttacaggtcccatctcttttgccttggcccttagaatatgct360

tctgaatcttcccagttgccgtcttcggtaatggtccaaatattacggactttggaaccc420

agtaagcaggcatcttctcccggcagaacttcattatatcttctgccaatcgtggctcat480

ctttatcggctccttgcttcaatgttatgaaagcacaaggagactctccccaacgctcat540

ctggcctagccaccacagaagcctcgagaattgcagggtgcaagtaaagtaagttttcta600

cctccaaactactgatgttttcacctccagagatgataatgtcctttgatctgtctttga660

tttctatgtagttatcgggatgctttacgccaagatcccctgaatgaaaccacccatctg720

caaaagcttcctcattggcttttgggttctttaagtagcccttcatcacactattaccac780

ccaacactatctctcctatggtttttccatcagcaggaacaggtttctttgttcgagtgt840

caatgacatctaaaaactctaaaccaatgtaccgtaccccttgacgtgaatttaggcgag900

cttgggtttcaggcggcagtgagttccactcaggcttccacgcacaaacagttgaaggac960

cataggtttctgagagaccatatgtgtgggtgacacggaagcccttctgggacattgaaa1020

agagaacagattggggtggagcggcaccagctgtcatcacatttacaacatgggggaggg1080

gaaggatagtgtcctctggtgaggcgttgactatggtgttgagaaccacaggtgcagcac1140

agaagtgagtcacaccatacttggctatggcagagtagactgccttggctgtcacctgcc1200

gaaggcatatgtttgtcccgcaaagagctgcaagtgaccaagtgtaacaccagccattgc1260

aatgaaacataggtaaagtccacaggtatacagctccttcattcaacccccatataagag1320

caccactcatagacattacatatgccccacggtgactcaacaccaccccctttggactgg1380

ctgttgtaccagaagtatatcctaaagcaatgctttgccactcatcctgtggtggcttcc1440

aagcaaattcagggtcacctgtttccaggaatttctcatactcaatggcacctcttccca1500

aagcatattccagtaccttaggatcacagctttcatcacctatgacaatcaaaattggag1560

gcttaaagttgcctttgcttttctcctccatgattttcaaagcttcttctgccacagaaa1620

aagactcttggtccaccataacaacagcagatgctgaatgacctagaaggaaggcaattg1680

tttgtgcatttagacgaatattgacactatttaacacagctccagccattggaactccaa1740

aatgagcttcatagagagccgggacatttggtgcaataacagctaccgtgctaccgagtc1800

caacagaccgtctagaaagagcagaggcgaacctacggcaacgctggtaagtctgatgcc1860

acgtgtagcgtacggatccgtggatcagggatgctctggaagggtggactgtagctgctc1920

tttctagaaaccagagcggcgtcaatgctgtataattcgccgcgttcttcggaagatcgt1980

ctatgtcgttccccgccgccatctctctggagcttcaccaccacctctaactctctatga2040

gttttagttttgtcaactagctgaagaaatgaaccaaaacgaaacctcaagtcg2094

序列2.鹽膚木rch2hd氨基酸序列（由轉(zhuǎn)錄組所得序列的反義鏈翻譯得到）

metalaalaglyasnaspileaspaspleuprolysasnalaala

151015

asntyrthralaleuthrproleutrppheleugluargalaala

202530

thrvalhisproserargalaserleuilehisglyservalarg

354045

tyrthrtrphisglnthrtyrglnargcysargargphealaser

505560

alaleuserargargservalglyleuglyserthrvalalaval

657075

ilealaproasnvalproalaleutyrglualahispheglyval

808590

prometalaglyalavalleuasnservalasnileargleuasn

95100105

alaglnthrilealapheleuleuglyhisseralaseralaval

110115120

valmetvalaspglngluserpheservalalagluglualaleu

125130135

lysilemetgluglulysserlysglyasnphelysproproile

140145150

leuilevalileglyaspglusercysaspprolysvalleuglu

155160165

tyralaleuglyargglyalaileglutyrglulyspheleuglu

170175180

thrglyaspprogluphealatrplysproproglnaspglutrp

185190195

glnserilealaleuglytyrthrserglythrthralaserpro

200205210

lysglyvalvalleuserhisargglyalatyrvalmetsermet

215220225

serglyalaleuiletrpglyleuasngluglyalavaltyrleu

230235240

trpthrleuprometphehiscysasnglytrpcystyrthrtrp

245250255

serleualaalaleucysglythrasnilecysleuargglnval

260265270

thralalysalavaltyrseralailealalystyrglyvalthr

275280285

hisphecysalaalaprovalvalleuasnthrilevalasnala

290295300

serprogluaspthrileleuproleuprohisvalvalasnval

305310315

metthralaglyalaalaproproglnservalleuphesermet

320325330

serglnlysglypheargvalthrhisthrtyrglyleuserglu

335340345

thrtyrglyproserthrvalcysalatrplysproglutrpasn

350355360

serleuproprogluthrglnalaargleuasnserargglngly

365370375

valargtyrileglyleuglupheleuaspvalileaspthrarg

380385390

thrlyslysprovalproalaaspglylysthrileglygluile

395400405

valleuglyglyasnservalmetlysglytyrleulysasnpro

410415420

lysalaasngluglualaphealaaspglytrpphehissergly

425430435

aspleuglyvallyshisproaspasntyrilegluilelysasp

440445450

argserlysaspileileileserglyglygluasnileserser

455460465

leugluvalgluasnleuleutyrleuhisproalaileleuglu

470475480

alaservalvalalaargproaspgluargtrpglygluserpro

485490495

cysalapheilethrleulysglnglyalaasplysaspglupro

500505510

argleualagluaspilemetlysphecysargglulysmetpro

515520525

alatyrtrpvalprolysservalilepheglyproleuprolys

530535540

thralathrglylysileglnlyshisileleuargalalysala

545550555

lysglumetglyprovallysargserlysleu

560565

序列3.鹽膚木rch2hd基因堿基序列與編碼氨基酸序列的對(duì)應(yīng)圖（此氨基酸序列為轉(zhuǎn)錄組得到序列的反義鏈所編碼，和正義鏈編碼的氨基酸序列一致）：

cgacttgaggtttcgttttggttcatttcttcagctagttgacaaaactaaaactcatag60

agagttagaggtggtggtgaagctccagagagatggcggcggggaacgacatagac116

metalaalaglyasnaspileasp

15

gatcttccgaagaacgcggcgaattatacagcattgacgccgctctgg164

aspleuprolysasnalaalaasntyrthralaleuthrproleutrp

101520

tttctagaaagagcagctacagtccacccttccagagcatccctgatc212

pheleugluargalaalathrvalhisproserargalaserleuile

25303540

cacggatccgtacgctacacgtggcatcagacttaccagcgttgccgt260

hisglyservalargtyrthrtrphisglnthrtyrglnargcysarg

455055

aggttcgcctctgctctttctagacggtctgttggactcggtagcacg308

argphealaseralaleuserargargservalglyleuglyserthr

606570

gtagctgttattgcaccaaatgtcccggctctctatgaagctcatttt356

valalavalilealaproasnvalproalaleutyrglualahisphe

758085

ggagttccaatggctggagctgtgttaaatagtgtcaatattcgtcta404

glyvalprometalaglyalavalleuasnservalasnileargleu

9095100

aatgcacaaacaattgccttccttctaggtcattcagcatctgctgtt452

asnalaglnthrilealapheleuleuglyhisseralaseralaval

105110115120

gttatggtggaccaagagtctttttctgtggcagaagaagctttgaaa450

valmetvalaspglngluserpheservalalagluglualaleulys

125130135

atcatggaggagaaaagcaaaggcaactttaagcctccaattttgatt548

ilemetgluglulysserlysglyasnphelysproproileleuile

140145150

gtcataggtgatgaaagctgtgatcctaaggtactggaatatgctttg596

valileglyaspglusercysaspprolysvalleuglutyralaleu

155160165

ggaagaggtgccattgagtatgagaaattcctggaaacaggtgaccct644

glyargglyalaileglutyrglulyspheleugluthrglyasppro

170175180

gaatttgcttggaagccaccacaggatgagtggcaaagcattgcttta692

gluphealatrplysproproglnaspglutrpglnserilealaleu

185190195200

ggatatacttctggtacaacagccagtccaaagggggtggtgttgagt740

glytyrthrserglythrthralaserprolysglyvalvalleuser

205210215

caccgtggggcatatgtaatgtctatgagtggtgctcttatatggggg788

hisargglyalatyrvalmetsermetserglyalaleuiletrpgly

220225230

ttgaatgaaggagctgtatacctgtggactttacctatgtttcattgc836

leuasngluglyalavaltyrleutrpthrleuprometphehiscys

235240245

aatggctggtgttacacttggtcacttgcagctctttgcgggacaaac884

asnglytrpcystyrthrtrpserleualaalaleucysglythrasn

250255260

atatgccttcggcaggtgacagccaaggcagtctactctgccatagcc932

ilecysleuargglnvalthralalysalavaltyrseralaileala

265270275280

aagtatggtgtgactcacttctgtgctgcacctgtggttctcaacacc980

lystyrglyvalthrhisphecysalaalaprovalvalleuasnthr

285290295

atagtcaacgcctcaccagaggacactatccttcccctcccccatgtt1028

ilevalasnalaserprogluaspthrileleuproleuprohisval

300305310

gtaaatgtgatgacagctggtgccgctccaccccaatctgttctcttt1076

valasnvalmetthralaglyalaalaproproglnservalleuphe

315320325

tcaatgtcccagaagggcttccgtgtcacccacacatatggtctctca1124

sermetserglnlysglypheargvalthrhisthrtyrglyleuser

330335340

gaaacctatggtccttcaactgtttgtgcgtggaagcctgagtggaac1172

gluthrtyrglyproserthrvalcysalatrplysproglutrpasn

345350355360

tcactgccgcctgaaacccaagctcgcctaaattcacgtcaaggggta1220

serleuproprogluthrglnalaargleuasnserargglnglyval

365370375

cggtacattggtttagagtttttagatgtcattgacactcgaacaaag1268

argtyrileglyleuglupheleuaspvalileaspthrargthrlys

380385390

aaacctgttcctgctgatggaaaaaccataggagagatagtgttgggt1316

lysprovalproalaaspglylysthrileglygluilevalleugly

395400405

ggtaatagtgtgatgaagggctacttaaagaacccaaaagccaatgag1364

glyasnservalmetlysglytyrleulysasnprolysalaasnglu

410415420

gaagcttttgcagatgggtggtttcattcaggggatcttggcgtaaag1412

glualaphealaaspglytrpphehisserglyaspleuglyvallys

425430435440

catcccgataactacatagaaatcaaagacagatcaaaggacattatc1460

hisproaspasntyrilegluilelysaspargserlysaspileile

445450455

atctctggaggtgaaaacatcagtagtttggaggtagaaaacttactt1508

ileserglyglygluasnileserserleugluvalgluasnleuleu

460465470

tacttgcaccctgcaattctcgaggcttctgtggtggctaggccagat1556

tyrleuhisproalaileleuglualaservalvalalaargproasp

475480485

gagcgttggggagagtctccttgtgctttcataacattgaagcaagga1604

gluargtrpglygluserprocysalapheilethrleulysglngly

490495500

gccgataaagatgagccacgattggcagaagatataatgaagttctgc1652

alaasplysaspgluproargleualagluaspilemetlysphecys

505510515520

cgggagaagatgcctgcttactgggttccaaagtccgtaatatttgga1700

argglulysmetproalatyrtrpvalprolysservalilephegly

525530535

ccattaccgaagacggcaactgggaagattcagaagcatattctaagg1748

proleuprolysthralathrglylysileglnlyshisileleuarg

540545550

gccaaggcaaaagagatgggacctgtaaagaggagtaagctgtaa1793

alalysalalysglumetglyprovallysargserlysleu＊

555560565

gtatgattctctgatgatgacgaaacagaaaacaggctcttggtcatgttatctttttga1853

ctttgtagaaaaattatgcttcatgggtatctccaataaatgttgcttgtaatatcataa1913

actatggacataaatagtttaagtggttattctgtctcctaaaagacttttaaatttctt1973

gagaaatgtcacaagttactttcgttttaagcttgggagaaagctgatatattgttgtgg2033

actaaaggttgagttctttgtatcaatgatgaagtagggccaagggccctaaaaattgtt2093

t2094。

序列1.鹽膚木rch2hd基因序列（2094bp）

aaacaatttttagggcccttggccctacttcatcattgatacaaagaactcaacctttag60

tccacaacaatatatcagctttctcccaagcttaaaacgaaagtaacttgtgacatttct120

caagaaatttaaaagtcttttaggagacagaataaccacttaaactatttatgtccatag180

tttatgatattacaagcaacatttattggagatacccatgaagcataatttttctacaaa240

gtcaaaaagataacatgaccaagagcctgttttctgtttcgtcatcatcagagaatcata300

cttacagcttactcctctttacaggtcccatctcttttgccttggcccttagaatatgct360

tctgaatcttcccagttgccgtcttcggtaatggtccaaatattacggactttggaaccc420

agtaagcaggcatcttctcccggcagaacttcattatatcttctgccaatcgtggctcat480

ctttatcggctccttgcttcaatgttatgaaagcacaaggagactctccccaacgctcat540

ctggcctagccaccacagaagcctcgagaattgcagggtgcaagtaaagtaagttttcta600

cctccaaactactgatgttttcacctccagagatgataatgtcctttgatctgtctttga660

tttctatgtagttatcgggatgctttacgccaagatcccctgaatgaaaccacccatctg720

caaaagcttcctcattggcttttgggttctttaagtagcccttcatcacactattaccac780

ccaacactatctctcctatggtttttccatcagcaggaacaggtttctttgttcgagtgt840

caatgacatctaaaaactctaaaccaatgtaccgtaccccttgacgtgaatttaggcgag900

cttgggtttcaggcggcagtgagttccactcaggcttccacgcacaaacagttgaaggac960

cataggtttctgagagaccatatgtgtgggtgacacggaagcccttctgggacattgaaa1020

agagaacagattggggtggagcggcaccagctgtcatcacatttacaacatgggggaggg1080

gaaggatagtgtcctctggtgaggcgttgactatggtgttgagaaccacaggtgcagcac1140

agaagtgagtcacaccatacttggctatggcagagtagactgccttggctgtcacctgcc1200

gaaggcatatgtttgtcccgcaaagagctgcaagtgaccaagtgtaacaccagccattgc1260

aatgaaacataggtaaagtccacaggtatacagctccttcattcaacccccatataagag1320

caccactcatagacattacatatgccccacggtgactcaacaccaccccctttggactgg1380

ctgttgtaccagaagtatatcctaaagcaatgctttgccactcatcctgtggtggcttcc1440

aagcaaattcagggtcacctgtttccaggaatttctcatactcaatggcacctcttccca1500

aagcatattccagtaccttaggatcacagctttcatcacctatgacaatcaaaattggag1560

gcttaaagttgcctttgcttttctcctccatgattttcaaagcttcttctgccacagaaa1620

aagactcttggtccaccataacaacagcagatgctgaatgacctagaaggaaggcaattg1680

tttgtgcatttagacgaatattgacactatttaacacagctccagccattggaactccaa1740

aatgagcttcatagagagccgggacatttggtgcaataacagctaccgtgctaccgagtc1800

caacagaccgtctagaaagagcagaggcgaacctacggcaacgctggtaagtctgatgcc1860

acgtgtagcgtacggatccgtggatcagggatgctctggaagggtggactgtagctgctc1920

tttctagaaaccagagcggcgtcaatgctgtataattcgccgcgttcttcggaagatcgt1980

ctatgtcgttccccgccgccatctctctggagcttcaccaccacctctaactctctatga2040

gttttagttttgtcaactagctgaagaaatgaaccaaaacgaaacctcaagtcg2094

序列2.鹽膚木rch2hd氨基酸序列（由轉(zhuǎn)錄組所得序列的反義鏈翻譯得到）

metalaalaglyasnaspileaspaspleuprolysasnalaala

151015

asntyrthralaleuthrproleutrppheleugluargalaala

202530

thrvalhisproserargalaserleuilehisglyservalarg

354045

tyrthrtrphisglnthrtyrglnargcysargargphealaser

505560

alaleuserargargservalglyleuglyserthrvalalaval

657075

ilealaproasnvalproalaleutyrglualahispheglyval

808590

prometalaglyalavalleuasnservalasnileargleuasn

95100105

alaglnthrilealapheleuleuglyhisseralaseralaval

110115120

valmetvalaspglngluserpheservalalagluglualaleu

125130135

lysilemetgluglulysserlysglyasnphelysproproile

140145150

leuilevalileglyaspglusercysaspprolysvalleuglu

155160165

tyralaleuglyargglyalaileglutyrglulyspheleuglu

170175180

thrglyaspprogluphealatrplysproproglnaspglutrp

185190195

glnserilealaleuglytyrthrserglythrthralaserpro

200205210

lysglyvalvalleuserhisargglyalatyrvalmetsermet

215220225

serglyalaleuiletrpglyleuasngluglyalavaltyrleu

230235240

trpthrleuprometphehiscysasnglytrpcystyrthrtrp

245250255

serleualaalaleucysglythrasnilecysleuargglnval

260265270

thralalysalavaltyrseralailealalystyrglyvalthr

275280285

hisphecysalaalaprovalvalleuasnthrilevalasnala

290295300

serprogluaspthrileleuproleuprohisvalvalasnval

305310315

metthralaglyalaalaproproglnservalleuphesermet

320325330

serglnlysglypheargvalthrhisthrtyrglyleuserglu

335340345

thrtyrglyproserthrvalcysalatrplysproglutrpasn

350355360

serleuproprogluthrglnalaargleuasnserargglngly

365370375

valargtyrileglyleuglupheleuaspvalileaspthrarg

380385390

thrlyslysprovalproalaaspglylysthrileglygluile

395400405

valleuglyglyasnservalmetlysglytyrleulysasnpro

410415420

lysalaasngluglualaphealaaspglytrpphehissergly

425430435

aspleuglyvallyshisproaspasntyrilegluilelysasp

440445450

argserlysaspileileileserglyglygluasnileserser

455460465

leugluvalgluasnleuleutyrleuhisproalaileleuglu

470475480

alaservalvalalaargproaspgluargtrpglygluserpro

485490495

cysalapheilethrleulysglnglyalaasplysaspglupro

500505510

argleualagluaspilemetlysphecysargglulysmetpro

515520525

alatyrtrpvalprolysservalilepheglyproleuprolys

530535540

thralathrglylysileglnlyshisileleuargalalysala

545550555

lysglumetglyprovallysargserlysleu

560565

序列3.鹽膚木rch2hd基因堿基序列與編碼氨基酸序列的對(duì)應(yīng)圖（此氨基酸序列為轉(zhuǎn)錄組得到序列的反義鏈所編碼，和正義鏈編碼的氨基酸序列一致）：

cgacttgaggtttcgttttggttcatttcttcagctagttgacaaaactaaaactcatag60

agagttagaggtggtggtgaagctccagagagatggcggcggggaacgacatagac116

metalaalaglyasnaspileasp

15

gatcttccgaagaacgcggcgaattatacagcattgacgccgctctgg164

aspleuprolysasnalaalaasntyrthralaleuthrproleutrp

101520

tttctagaaagagcagctacagtccacccttccagagcatccctgatc212

pheleugluargalaalathrvalhisproserargalaserleuile

25303540

cacggatccgtacgctacacgtggcatcagacttaccagcgttgccgt260

hisglyservalargtyrthrtrphisglnthrtyrglnargcysarg

455055

aggttcgcctctgctctttctagacggtctgttggactcggtagcacg308

argphealaseralaleuserargargservalglyleuglyserthr

606570

gtagctgttattgcaccaaatgtcccggctctctatgaagctcatttt356

valalavalilealaproasnvalproalaleutyrglualahisphe

758085

ggagttccaatggctggagctgtgttaaatagtgtcaatattcgtcta404

glyvalprometalaglyalavalleuasnservalasnileargleu

9095100

aatgcacaaacaattgccttccttctaggtcattcagcatctgctgtt452

asnalaglnthrilealapheleuleuglyhisseralaseralaval

105110115120

gttatggtggaccaagagtctttttctgtggcagaagaagctttgaaa450

valmetvalaspglngluserpheservalalagluglualaleulys

125130135

atcatggaggagaaaagcaaaggcaactttaagcctccaattttgatt548

ilemetgluglulysserlysglyasnphelysproproileleuile

140145150

gtcataggtgatgaaagctgtgatcctaaggtactggaatatgctttg596

valileglyaspglusercysaspprolysvalleuglutyralaleu

155160165

ggaagaggtgccattgagtatgagaaattcctggaaacaggtgaccct644

glyargglyalaileglutyrglulyspheleugluthrglyasppro

170175180

gaatttgcttggaagccaccacaggatgagtggcaaagcattgcttta692

gluphealatrplysproproglnaspglutrpglnserilealaleu

185190195200

ggatatacttctggtacaacagccagtccaaagggggtggtgttgagt740

glytyrthrserglythrthralaserprolysglyvalvalleuser

205210215

caccgtggggcatatgtaatgtctatgagtggtgctcttatatggggg788

hisargglyalatyrvalmetsermetserglyalaleuiletrpgly

220225230

ttgaatgaaggagctgtatacctgtggactttacctatgtttcattgc836

leuasngluglyalavaltyrleutrpthrleuprometphehiscys

235240245

aatggctggtgttacacttggtcacttgcagctctttgcgggacaaac884

asnglytrpcystyrthrtrpserleualaalaleucysglythrasn

250255260

atatgccttcggcaggtgacagccaaggcagtctactctgccatagcc932

ilecysleuargglnvalthralalysalavaltyrseralaileala

265270275280

aagtatggtgtgactcacttctgtgctgcacctgtggttctcaacacc980

lystyrglyvalthrhisphecysalaalaprovalvalleuasnthr

285290295

atagtcaacgcctcaccagaggacactatccttcccctcccccatgtt1028

ilevalasnalaserprogluaspthrileleuproleuprohisval

300305310

gtaaatgtgatgacagctggtgccgctccaccccaatctgttctcttt1076

valasnvalmetthralaglyalaalaproproglnservalleuphe

315320325

tcaatgtcccagaagggcttccgtgtcacccacacatatggtctctca1124

sermetserglnlysglypheargvalthrhisthrtyrglyleuser

330335340

gaaacctatggtccttcaactgtttgtgcgtggaagcctgagtggaac1172

gluthrtyrglyproserthrvalcysalatrplysproglutrpasn

345350355360

tcactgccgcctgaaacccaagctcgcctaaattcacgtcaaggggta1220

serleuproprogluthrglnalaargleuasnserargglnglyval

365370375

cggtacattggtttagagtttttagatgtcattgacactcgaacaaag1268

argtyrileglyleuglupheleuaspvalileaspthrargthrlys

380385390

aaacctgttcctgctgatggaaaaaccataggagagatagtgttgggt1316

lysprovalproalaaspglylysthrileglygluilevalleugly

395400405

ggtaatagtgtgatgaagggctacttaaagaacccaaaagccaatgag1364

glyasnservalmetlysglytyrleulysasnprolysalaasnglu

410415420

gaagcttttgcagatgggtggtttcattcaggggatcttggcgtaaag1412

glualaphealaaspglytrpphehisserglyaspleuglyvallys

425430435440

catcccgataactacatagaaatcaaagacagatcaaaggacattatc1460

hisproaspasntyrilegluilelysaspargserlysaspileile

445450455

atctctggaggtgaaaacatcagtagtttggaggtagaaaacttactt1508

ileserglyglygluasnileserserleugluvalgluasnleuleu

460465470

tacttgcaccctgcaattctcgaggcttctgtggtggctaggccagat1556

tyrleuhisproalaileleuglualaservalvalalaargproasp

475480485

gagcgttggggagagtctccttgtgctttcataacattgaagcaagga1604

gluargtrpglygluserprocysalapheilethrleulysglngly

490495500

gccgataaagatgagccacgattggcagaagatataatgaagttctgc1652

alaasplysaspgluproargleualagluaspilemetlysphecys

505510515520

cgggagaagatgcctgcttactgggttccaaagtccgtaatatttgga1700

argglulysmetproalatyrtrpvalprolysservalilephegly

525530535

ccattaccgaagacggcaactgggaagattcagaagcatattctaagg1748

proleuprolysthralathrglylysileglnlyshisileleuarg

540545550

gccaaggcaaaagagatgggacctgtaaagaggagtaagctgtaa1793

alalysalalysglumetglyprovallysargserlysleu＊

555560565

gtatgattctctgatgatgacgaaacagaaaacaggctcttggtcatgttatctttttga1853

ctttgtagaaaaattatgcttcatgggtatctccaataaatgttgcttgtaatatcataa1913

actatggacataaatagtttaagtggttattctgtctcctaaaagacttttaaatttctt1973

gagaaatgtcacaagttactttcgttttaagcttgggagaaagctgatatattgttgtgg2033

actaaaggttgagttctttgtatcaatgatgaagtagggccaagggccctaaaaattgtt2093

t2094