利用DDRT-PCR鑒定蕓芥自交不親和系與自交親和系的差異表達cDNA

范惠玲，白生文，李華清，孫萬倉

（1.河西學院農(nóng)業(yè)與生物技術(shù)學院，甘肅張掖 734000；2.甘肅農(nóng)業(yè)大學農(nóng)學院，甘肅蘭州 730070）

利用DDRT-PCR鑒定蕓芥自交不親和系與自交親和系的差異表達cDNA

范惠玲1，白生文1，李華清1，孫萬倉2

（1.河西學院農(nóng)業(yè)與生物技術(shù)學院，甘肅張掖 734000；2.甘肅農(nóng)業(yè)大學農(nóng)學院，甘肅蘭州 730070）

蕓芥與蕓薹屬植物具有親緣關(guān)系，也是蕓薹屬植物的重要育種資源。自交親和性是蕓芥的一種重要變異性狀。為了探明蕓芥自交親和基因的表達器官，并分離與自交親和性有關(guān)的cDNA片段，以蕓芥的一對近等基因系，自交親和系（SC）與自交不親和系（SI）為試材，利用差異顯示RT-PCR技術(shù)分別對開花前和開花后的葉片、花藥和柱頭進行鑒定。結(jié)果表明，不論開花前還是開花后，蕓芥自交親和系與自交不親和系的葉片或花藥間的擴增帶型是一樣的，但在近等基因系的柱頭間得到了差異表達條帶。這證明蕓芥自交親和基因不是組成型表達，而是組織特異性表達，柱頭是其唯一的表達器官。在開花前的自交親和系中共得到了2條cDNA片段，一條小于100 bp，另一條為750～1 000 bp，在開花后的自交親和系中得到了1條300 bp的cDNA片段。據(jù)分析這3條cDNA片段可能與蕓芥的自交親和性密切相關(guān)。在開花前的自交不親和系中共得到了2條cDNA片段，一條為500 bp，另一條為750 bp，同時在開花后的自交不親和系中得到了1條500～600 bp的cDNA片段。這些cDNA片段可以用來區(qū)分蕓芥的自交親和系與自交不親和系，還可用于克隆自交親和基因。

蕓芥；自交親和系；自交不親和系；DDRT-PCR

Yunjie（Eruca sativa）Mill.is known as rocket，rocket salad，roquette，arugula，taramira，or rocala in the West［1-3］.The most common name of E.sativa in China is Yunjie.In other parts of the world，it ismostly used a leafy vegetable crop and herbal medicine.In China and India，Yunjie is cultivated as an oilseed crop.It is known for its high tolerance to soil infertility，saline-alkaline and cold injury［4］，as well as its resistance to drought and powdery m ildew［5］.Yunjie is a highly self-incompatible（SI）plant and its population are made up of individuals with different genotypes. Therefore，the yield potential is restricted by heterozygosity［6］.Previous research has shown that，by open pollination，the seed rate of self-compatible（SC）plant is higher than that of SI plant［7］.When hybrid seeds were produced using male-sterile line，SC lines were used as the maintainer line of sterile，then the cost of seed production might be reduced，and yield of hybrid seeds could also be increased［8］.Yamane et al［9］found that，to a certain extent，SC could shorten the time of plant breeding.Studies on SC in plants can be used to clarify genetic mechanism of SC in SI plants，evolutionary relationship SI and SC，and interaction mechanism of pollen-pistil.Therefore，studies on SC in SI plants are of great significance for the biology and p lant breeding.

Deletion of gene fragments underlying self-incompatibility may cause SC in SI p lants［10］.He et al［11］reported that partial deletion of protein coded by S_locus glycoprotein（SLG）and extra-cellular S_locus receptor kinase（eSRK）led to self-compatibility of Qinghai big yellow rapeseed（B.campestris）and Yellow sarson（B. campestris）.Schopfer et al［12］found that gene deletion of S_locus cysteine rich（SCR）and S_locus protein11（SP11）caused SC of B.campestris.Nasrallah et al［13］suggested that self-compatible line in B.oleracea with S-f1（self-compatible gene）was lack of S_locus receptor kinase（SRK）.There has been some progress on studies on self-compatibility of Yunjie.For example，the activity of SOD，POD and CAT in SIand SC lines were determined by Wang et al［14］，the results showed that regulation of self-compatible genes in SC line was related to enzymes activity.Chen et al［15］showed that the self-compatibility regulation of Yunjie was in connection with Xyloglucan endotransglycosylase/hydrolase gene.Fang et al［16］cloned the DnaJ homolog gene from Eruca sativa.In this study，DDRT-PCR（Differential disp lay reverse transcriptase polymerase chain reaction）was used to isolate gene fragments related to selfcompatibility from SI and SC lines in Yunjie.The results would provide some theoretical and technical support for further studies on formation mechanism of selfcompatibility of Yunjie.

1 Materials and methods

1.1 Plant m aterials

We used a pair of near isogenic lines（NILs）of Yunjie，self-incompatible（SI）and self-compatible（SC）lines.SC line was amutant from SI line，and selected after examination of several morphological and agronomic traits.

As a result of five generations of forced bud-pollination，SI line has stable self-incompatibility and SC line has stable self-compatibility.Seed was obtained from the Gansu Agricultural University，Lanzhou，China.Plants were grown at field conditions.Capitula were covered in the bud stage with sulfate paper bags，leaves，anthers and stigmas were collected from SI and SC plants before flowering（5 mm buds）and after flowering（opened buds），frozen in liquid nitrogen prior to store at-80℃.

1.2 Experim entm ethods

1.2.1 Extraction and preparation of RNA Total RNA extraction from each line was conducted according to the guanidinium isothiocyanatemethod described by Wang［17］and modified by Golldack et al［18］.Before cDNA synthesis，the RNA sample was treated with RNase-free DNAase（Promega，Toulouse，F(xiàn)rance）for 1 h at 37℃，to elim inate any contam ination with genomic DNA.For absorbance ratio in OD260/280and concentration analyses the Nanophotometer Nanogram（IMPLEN，Germany）was used，and the integrity was assayed in denatured agarose electrophoresis.The obtained RNA samples were diluted with 1×TE solution and stored at-80℃for later use.

1.2.2 Design and preparation of primers Based on the characteristic of primer used in DDRT-PCR and research report by Yan［19］and Yu［20］，three anchor primers with 3′end（H-T11M）and ten 10 bp random primers were designed and synthesized by Bao Biological Engineering Co.，Ltd.（TaKaRa Inc）（Tab.1）. Each primer was diluted to 100μmol/L preservation solution，then working solution of anchor and random primers were diluted again 10-fold and 20-fold，respectively.

Tab.1 Sequences of anchor and random prim ers

1.2.3 Synthesis of the first chain of cDNA 13.4 μL mixture composed of templates RNA and anchor primer（2.27μL of 10μmol/L H-T11M，2μL RNA，9.13μL DEPC-H2O）were prepared in Microtube，then placed in a water-bath at 70℃for 5 m in，rapid ly cooled in an ice water bath for 2 min；Denaturing solution of templates RNA/anchor primer was obtained by centrifugation for a while，then added to reaction solution used to reverse transcription（5μL of 5×M-MLV reaction Buffer，5μL of2.5 mmol/L dNTPs，0.6μL of 40 U/μL Ribonuclease inhibitor，1μL M-MLV reverse transcriptase（Promega），put in a water bath at 42℃for 60 m in，followed at 95℃for 5 m in to inactivate MMLV reverse transcriptase.

1.2.4 DDRT-PCR amp lification Reaction system was 25μL total volume contained 17.2μL DEPC-ddH2O，1.5μL 10×PCR Buffer（plus Mg2+），2.0 μL of 2.5 mmol/L dNTPs，1.0μL of 10μmol/L anchor primer，1.0μL of 5μmol/L random primer，1.5 μL samples of the single-stranded reaction，0.8μL of 2.5 U/μL Taq DNA polymerase.The reaction mixture was overlain with 10μL petroleum.The PCR reactions were performed in an Eppendorf Master Cycler Gradient（Eppendorf，Germany）：denaturation at 94℃for 2 m in；35 cycles of denaturation at 94℃for 30 s，annealing at 42℃for 1 min 30 s and extension at72℃for 1 m in；followed by a final extension at 72℃for 10 min.Selective amplification products were denatured in 50%formam ide at 95℃and separated by electrophoresis on an agarose gel（1.2%）containing urea and TAE.Bands were stained with ethidium brom ide and visualized and quantified with an image analyzer（Biorad，GelDoc XRS，F(xiàn)rance）.

2 Results and analysis

2.1 The am p lification pattern of leaf w ere the sam e in SI and SC lines of Yunjie

Leaves from SIand SC lines of Yunjie before flowering and after flowering，respectively，were analyzed by DDRT-PCR.By comparing of difference in leaf profiles between SI and SC lines，the amplified results showed that the same bands were exhibited in SI and SC lines with eighteen of the thirty primer combinations studied. When amplification reaction was performed with anchor and random primer of H-T11A/④and H-T11A/⑥，respectively，a fragment of 250-500 bp was amp lified in SIand SC lines before flowering（Fig.1）.When amplification reaction was carried out with anchor and random primer of H-T11G/⑧and H-T11C/⑤，respectively，one fragment of 500 bp was present both in SI and SC lines after flowering（Fig.2-3）.

Fig.1 Different disp lay profiles from leavesof SC and SI lines before flow ering

Fig.2 Different d isp lay p rofiles from leaves of SC and SI lines after flowering w ith p rim ers of H-T11G and⑧

Fig.3 Different d isp lay p rofiles from leaves of SC and SI lines after flowering w ith p rim ers of H-T11C and⑤

Fig.4 Differential disp lay p rofiles from anthers of SC and SI lines before flowering w ith prim ers of H-T11A/①

2.2 The am p lification pattern of an ther were the sam e betw een SI and SC lines of Yun jie

Gene fragments involved in the self-compatibility and self-incompatibility in Yunjie were identified by DDRT-PCR in anthers before flowering and after flowering，respectively.By comparing of difference in anthers from SIand SC lines，the amp lified results indicated that no differentially expressed bands were presented both in SIand SC lineswith different primer combinations.As shown in the example of the obtained expression profile（Fig.4），when amplification reaction was performed with anchor and random primer of HT11A and①，a fragment which is slightly higher than 250 bp was amplified both in SI and SC lines before flowering.When amplification reaction was carried out with anchor and random primer of H-T11C and③，two the same fragments were presented both in SI and SC lines after flowering，one was 250-500 bp，the other was 100-250 bp（Fig.5）.Seen from Fig.6，when amplification reaction was performed with anchor and random primer of H-T11A and②，a fragment of250-500 bp was displayed both in SIand SC lines after flowering.

Fig.5 Differential disp lay p rofiles from anthers of SC and SI lines after flow ering w ith p rim ers H-T11C/③

Fig.6 Differential disp lay profiles from anthers of SC and SI lines after flow ering w ith p rim ers H-T11A/②

2.3 The am p lification pattern of stigm as before flowering were different between SI and SC lines

Stigmas from SI and SC lines of Yunjie before flowering were analysised by DDRT-PCR.By comparing of difference in stigmas between SI and SC lines，the amplified results suggested that about 142 cDNA fragments were amplified with eighteen pairs of effective primer of thirty primer combinations studied.Fiftytwo amplification bands of which were generated in SI lines，and ninety cDNA bands were showed in SC lines.The length of the amplified cDNA fragments varied from 100 to 2 000 bp，depending on the primer combination used，mainly about 200-1 500 bp；Selfcompatible lines had ten differentially expressed bands，only two bands of which exhibited repeatedly with different pairs of primers，the one was less than 100 bp（Fig.7），the other was 750-1 000 bp（Fig.8）；In addition，six cDNA fragments were present in selfincompatible lines，while absent in self-compatible lines，of which two bands could be generated repeatedly，one was 500 bp（Fig.8），the other was slightly higher than 750 bp（Fig.9）.The results also indicated that total number of cDNA bands amplified with anchor H-T11C was higher than thatwith anchor H-T11A and H-T11G.

Fig.7 Differential disp lay profiles from stigm as of SC and SI lines before flowering w ith p rim ers H-T11C/④

Fig.8 Differential disp lay p rofiles from stigm as of SC and SI lines before flowering w ith p rim ers of H-T11G/②

Fig.9 Differential disp lay profiles from stigm as of SC and SI lines before flowering w ith p rim ers H-T11A/⑩

2.4 The am p lification pattern of stigm as after flowering were different in SI and SC lines

W ith eighteen pairs of effective primer，DDRTPCR on stigmas from SI and SC lines after flowering were conducted.The results showed that 133 fragments were obtained in PCR products，of which 87 ones were exhibited in SC lines and 46 ones in SI lines.The bands were mainly about 200-1 500 bp.Besides two specific bands which existed in SC lines before flowering，a differential band of 300 bp was found unique to SC lines after flowering（Fig.10）.Meanwhile，one band of 500-600 bp was identified in SI lines after flowering（Fig.11）.Above all，these results proved that three repetitive differential gene fragments were presented in stigmas of SC lines，which m ight be closely related to the self-compatible genes in Yunjie.

Fig.10 Profile of electrophoretic ream p lified cDNA from stigm as of SC and SI lines after flowering in 1.2%agarose gel using the p rim ers com bined w ith H-T11A and①

Fig.11 Profile of electrophoretic ream p lified cDNA from stigm as of SC and SI lines after flow ering in 1.2%agarose gel using the prim ers com bined w ith H-T11C and⑨，⑩

3 Discussion

Based on the expression time and tissue specificity of self-compatibility，SLG has been identified and isolated in stigma papilla cells from B.oleracea and B. campestris by immunohistochem istry method［21］.Immunohistochem istry and hybridization in situ showed that SLG accumulated in large quantities on the cell wall of papilla cells in mature stigma，but it did not exist in nutritive tissue，such as leaf，root，as well as in style，ovary and young seedling［22］.The expression time of SLG was in relation to that of self-incompatibility，when a plant express self-incompatibility，the content of SLG in stigma could reach 5%of total protein in stigma［22］. The molecular progress on the study of plant self-incompatibility also suggested that self-compatibility was associated with the deletion of self-incompatible gene fragments［15］.In our work，analysis on DDRT-PCR differential display found that when mRNA in leaves and anthers from SC and SI lines before and after flowering were amplified，differential bands were absent between SC and SI lines.This experiment result showed that self-compatible gene of Yunjie did not express in leaf and anther.However，when mRNA in stigma from SC and SI lines before and after flowering were amp lified with different primer combinations，some differential fragments were present in SC and SI lines.These results proved that self-compatible genes of E.sativa was not constitutive expression but tissue-specific expression，stigma was the only expression tissue.

The use of DDRT-PCR has often been described as an efficientmethod for studies on differential expression of cells or tissues under stress conditions，regulatory mechanism involved in vital processes occurring in living organisms，new genes isolation and cloning［23］. Up to now，this technique has been used widely in isolation of the stress resistant gene，disease resistance gene［24-25］，transgenic breeding［26］，heterosis mechanism［27］，etc.It also provides rapid and multiple comparisons of plant responses to stress durations［28］.

We used the sigmas of SI and SC lines in Yunjie before and after flowering as materials，differential expressed genes fragments were isolated with different primer combinations of anchor and random primers. The results of DDRT-PCR showed that six differential bands were obtained，of which three were present in SI lines，and three were exhibited in SC lines.Three PCR fragments of 300，100，750-1 000 bp may be closely connected with self-compatibility of Yunjie.On the one hand，SC line is a new self-compatible mutant except for Brassica genus，its self-compatibility was stable and high.Because SC line was in high level of self-compatibility.So that differential fragments were closely relation to self-compatibility of Yunjie.On the other hand，we used the self-incompatible lines as control（CK）in our experiments.CK has stable and high self-incompatibility，its self-compatible index was always less than one when pretreated after 7-8 generations of selfing. So，the SI line was highly self-incompatible.As a consequence，differential bands that were not relation to self-compatibility could be excluded.Furthermore，mRNA samples from the same growth period were amp lified with as many as possible primer combinations，each mRNA sample was amp lified in three rep lications，all these could ensure the dependability of the experiment results.

In conclusion，self-compatible genes of Yunjie was not constitutive expression but tissue-specific expression，stigma was the only expression tissue.Three reproducible cDNA fragments of 300，100，750-1 000 bp were presented in stigmas of SC lines，these cDNA fragments could efficiently distinguish self-compatibility and were useful for cloning self-compatible gene of Yunjie.

Acknow ledgem ent

The authors gratefully acknow ledge Ph D Ya-Juan ZHANG and Rob Ferguson，for the valuable comments on the manuscript.

［1］ Chen K，Zhang X B，Jiang J L.Plantlet regeneration from cotyledon，cotyledon petiole，and hypocotyl explants via somatic embryogenesis pathway in roquette（Eruca sativa）［J］.Plant Biosystems，2011，1-2：68-76.

［2］ Menéndez A I，Romero A M，F(xiàn)olcia A M.Aphid and episodic O3 injury in arugula plants grown in open-top field chambers［J］.Agriculture，Ecosystems and Environment，2010（1/2）：10-14.

［3］ Michalis O，Chara P，Dimitra K.Relationships between nitrogen，dry matter accumulation and glucosinolates in Eruca sativa Mill［J］.Plant and Soil，2012，1：347-358.

［4］ Fan H L，Luo F F，Huang X L，et al.Preliminary studies of the variation in self-compatibility among 52 cultivars of Eruca sativa［J］.Chinese Bulletin of Botany，2015，50（5）：598-604.

［5］ Sun W C，Pan Q Y，Liu Z G，et al.Overcoming self-incompatibility in Eruca sativa by chem ical treatment of stigmas［J］.Plant Genetic Resources，2005，1：13-18.

［6］ Fan H L，Bai SW，Chang L G，et al.Genetic variation of self-compatibility in Eruca sativa M ill.［J］.Chinese Journal of Oil Crop Sciences，2015，37（5）：605-613.

［7］ Coulibaly I，Noirot M，Loricux M，et al.Introgression of self-compatibility from Coffea heterocalyx to the cultivated species Coffea canephora［J］.Theoretical and Applied Genetics，2002，105（617）：994-999.

［8］ Fang Z Y，Liu Y M，Yang L M，et al.An important change in seed production techniques of the cabbage［J］.China Vegetables，2004，5：33.

［9］ Yamane H，Ushijima K，Sassa H，et al.The use of the S haplotype-specific F-box protein gene，SFB，as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot（Prunus mume）［J］.Theoretical and Applied Genetics，2003，107（8）：1357-1361.

［10］ Goring D R，Glavin T L，Schafer U，et al.An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion［J］.The Plant Cell，1993，5（5）：531-539.

［11］ He Y T，Ma C Z，Ma Y，et al.Cloning of S-locus gene by PCR walking in Brassica campestris L.［J］.Chinese Journal of Oil Crop Sciences，2004，26（1）：1-5.

［12］ Schopfer C R，Nasrallah M E，Nasrallah J B.The male determinant of self-incompatibility in Brassica［J］.Sci-ence，1999，286（5445）：1697-1700.

［13］ Nasrallah J B，Rundle S J，Nasrallah M E.Genetic evidence for the requirement of the Brassica S_locus receptor kunase gene in the self-incompatibility response［J］. Plant J，1994，5：373-384.

［14］ Wang B C，Sun W C，F(xiàn)an H L，et al.Studies on SOD，POD and CAT activity between self-compatible and selfincompatible lines in Eruca sativa M ill.［J］.Chinese Journal of Oil Crop Sciences，2006，28（2）：162-165.

［15］ Chen JR，F(xiàn)ang Y，Sun W C，et al.Cloning and bioinformatics of xyloglucan endotrans glycosylase and hydrolase Es XTH1 gene in Eruca sativa［J］.Chinese Journal of Oil Crop Sciences，2013，35（2）：131-136.

［16］ Fang Y，Yang G，Sun W C，et al.Cloning and expression of DnaJ homolog gene from Eruca sativa［J］.Acta Agriculturae Boreali-sinica，2015，30（6）：61-65.

［17］ Wang G L.Plant genetic engineering［M］.Beijing：Science Press，1998：608-609.

［18］ Golldack D，Su Hua，Quigley F，et al.Characterization of a HKT-type transporter in rice as a general alkali cation transporter［J］.The Plant Journal，2002，31（4）：529-542.

［19］ Yan H F.mRNA differential expression from interaction of puccinia recondita with Lr38 based on DDRT-PCR［D］.Baoding：Agricultural University of Hebei，2003.

［20］ Yu J.Isolation of genes related to K562 cells differentiation suing a modified DDRT-PCR［D］.Chongqing；Chongqing Medical University，2003.

［21］ Yua H F，Zhao Z Q，Sheng X G，et al.Identification of S-hap lotypes in DH-lines of broccoli（Brassica oleracea L.）［J］.Journal Of Horticultural Science and Biotechnology.2014，89（4）：430-434.

［22］ Fan H L，Wang W Q，Li Y L，et al.Study on S alleles and their expression in self-incompatible lines of Eruca Sativa Mill.［J］.Agricultural Research in the Arid Areas，2013，31（5）：110-116.

［23］ Ding C H，Du X W，Ying X，et al.Screening for differentially expressed genes in endophytic fungus strain 39 during co-culture with herbal extract of its host dioscorea nipponica makino［J］.Curr M icrobiol，2014，69（4）：517-524.

［24］ Hannappel U，Balzer H J and Ganal N W.Direct isolationof cDNA sequences from specific chromosomal regions of the tomato genome by the differential display technique［J］.Mol Gen Genet，1995，249：19-24.

［25］ Mei JF，Tang C Q，Xu D L，et al.Study on diggerential gene expression of tea plant（Camellia sinensis）during cold acclimation in winter［J］.Chinese Journal of Tropical Crops，2011，32（4）：648-652.

［26］ Liu H，Hu J，Pan L，et al.Robust ordered mRNA differential display：an improved method for global gene expression profiling［J］.Biotechniques，2011，51（4）：271-275.

［27］ Ni Z F，Sun Q X，Liu Z Y.Differential gene expression between heterotie and nonheterotic wheat hybrids and their parental inbreds in seedling leaves［J］.Journal of Agricultural Biotechnology，1999，7（1）：95-100.

［28］ Sadia F，Amer J.Expression profiling of bioactive genes from amedicinal plant Nigella sativa L［J］.Applied Biochemistry and Biotechnology，2013，170（6）：1472-1481.

Identification of cDNA Fragm ents in Self-incom patible and Self-com patible Lines of Eruca sativa M ill.by DDRT-PCR

FAN Huiling1，BAIShengwen1，LIHuaqing1，SUN Wancang2
（1.College of Agriculture and Biotechnology，Hexi University，Zhangye 734000，China；2.College of Agronomy，Gansu Agricultural University，Lanzhou 730070，China）

Yunjie is closely related to important vegetables and oil seed crops，and can be considered a genetic resource for all Brassiceae crops.Self compatibility is an important variant traits to Yunjie.This study aimed to probe the expression organ of self-compatible gene and isolate cDNA fragments related to self-compatibility of Yunjie. Leaf，anther and stigma from self-incompatible（SI）and self-compatible（SC）lines of Yunjie were exam ined by DDRT-PCR.The results showed that the amplification pattern of leaves and anthers were the same in SI and SC lines.However，different bandswere obtained in stigmas of SIand SC lines before and after flowering.SC gene of E. sativa was not showing constitutive expression but showed tissue-specific expression in stigma.Only two fragments were amplified in SC lines before flowering，the one was less than 100 bp，the other was 750-1 000 bp.A 300 bp fragment was found unique to self-compatible line after flowering.These three cDNA fragmentsm ight be closely related to the self-compatibility in Yunjie.In addition，two cDNA fragmentswere generated in SI line before flowering，one was 500 bp，the other was 750 bp.One band of 500-600 bp was identified in SI line after flowering.These cDNA fragments could efficiently distinguish self-compatible and self-incompatible lines，and could also be usefulfor cloning self-compatible gene of Yunjie.

Yunjie；Self-compatibility；Self-incompatibility；DDRT-PCR

Q78 文獻標識碼：A 文章編號：1000-7091（2016）03-0044-07

10.7668/hbnxb.2016.03.007

2016-03-19

甘肅省高等學?？蒲许椖浚?015A-136）

范惠玲（1980-），女，甘肅隴西人，講師，碩士，主要從事油菜近緣植物的遺傳和育種研究。