利用DDRT-PCR鉴定芸芥自交不亲和系与自交亲和系的差异表达cDNA

范惠玲，白生文，李华清，孙万仓

（1.河西学院农业与生物技术学院，甘肃张掖 734000；2.甘肃农业大学农学院，甘肃兰州 730070）

利用DDRT-PCR鉴定芸芥自交不亲和系与自交亲和系的差异表达cDNA

范惠玲1，白生文1，李华清1，孙万仓2

（1.河西学院农业与生物技术学院，甘肃张掖 734000；2.甘肃农业大学农学院，甘肃兰州 730070）

芸芥与芸薹属植物具有亲缘关系，也是芸薹属植物的重要育种资源。自交亲和性是芸芥的一种重要变异性状。为了探明芸芥自交亲和基因的表达器官，并分离与自交亲和性有关的cDNA片段，以芸芥的一对近等基因系，自交亲和系（SC）与自交不亲和系（SI）为试材，利用差异显示RT-PCR技术分别对开花前和开花后的叶片、花药和柱头进行鉴定。结果表明，不论开花前还是开花后，芸芥自交亲和系与自交不亲和系的叶片或花药间的扩增带型是一样的，但在近等基因系的柱头间得到了差异表达条带。这证明芸芥自交亲和基因不是组成型表达，而是组织特异性表达，柱头是其唯一的表达器官。在开花前的自交亲和系中共得到了2条cDNA片段，一条小于100 bp，另一条为750～1 000 bp，在开花后的自交亲和系中得到了1条300 bp的cDNA片段。据分析这3条cDNA片段可能与芸芥的自交亲和性密切相关。在开花前的自交不亲和系中共得到了2条cDNA片段，一条为500 bp，另一条为750 bp，同时在开花后的自交不亲和系中得到了1条500～600 bp的cDNA片段。这些cDNA片段可以用来区分芸芥的自交亲和系与自交不亲和系，还可用于克隆自交亲和基因。

芸芥；自交亲和系；自交不亲和系；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，France）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，France）.

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.

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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 文献标识码：A 文章编号：1000-7091（2016）03-0044-07

10.7668/hbnxb.2016.03.007

2016-03-19

甘肃省高等学校科研项目（2015A-136）

范惠玲（1980-），女，甘肃陇西人，讲师，硕士，主要从事油菜近缘植物的遗传和育种研究。