آنالیز کارکرد ژن Fol-SIX1 در ارقام مقاوم و حساس Cucumis melo

نوع مقاله: علمی پژوهشی

نویسندگان

1 دانشجوی کارشناسی‌ارشد بیوتکنولوژی کشاورزی دانشکده کشاورزی، دانشگاه فردوسی مشهد

2 استادیار پژوهشکده علوم گیاهی، دانشگاه فردوسی مشهد

3 استادیار گروه بیوتکنولوژی و به‌نژادی گیاهان زراعی دانشکده کشاورزی، دانشگاه فردوسی مشهد

4 استاد گروه بیوتکنولوژی و به‌نژادی گیاهان زراعی دانشکده کشاورزی، دانشگاه فردوسی مشهد

چکیده

ژن‎های اثرگذار (effector genes) نقش کلیدی را در برهم‌کنش میان بیمارگر و گیاه ایفا می‌کنند. جستجوی همولوگ ژن‎های اثرگذار از زمان گزارش تعدادی از این ژن‎های در فرم اختصاصی Fusarium oxysporum f. sp. lycopersici (Fol) در دیگر فرم‌های اختصاصی آغاز شده است. اخیراً، با بهره‌گیری از روش‌های مبتنی بر بیوانفورماتیک همولوگ ژن اثرگذار Fol-SIX1 از فرم اختصاصیFusarium oxysporum f. sp. melonis (Fom) گزارش شده‌است. در این مطالعه آنالیز عملکرد ژن Fol-SIX1 با هدف بررسی برهمکنش این ژن با ارقام افتراقی ملون حامل ژن های مقاومت، انجام شد. توالی کدکننده ژن Fol-SIX1 از سازه pTS1 طی سه مرحله در وکتور جفتی pCAMBIA3301 همسانه‌سازی شد و با استفاده از روش های کلنی PCR و هضم آنزیمی تایید گردید. صحت توالی سازه بیانی pCaS1 با توالی‌یابی دوجهته بوسیله آغازگرهای PSh4-F2  و  PSh51-Rمورد بررسی قرار گرفت. روش بیان موقت مبتنی بر تزریق اگروباکتریوم برای بیان ژن Fol-SIX1 در برگ ارقام ملون استفاده شد. آنالیز کارکرد ژن Fol-SIX1 با تزریق سویه LBA4404 حامل سازه بیانی pCaS1 در برگ ارقام ملون انجام شد. برگ ارقام  Charentais T (Ch-T)و Charentais Fom2 (Ch-Fom2) پس گذشت 24 ساعت در ناحیه تزریق علائم سبزخشکی ناشی از مرگ سلولی در پاسخ به بیان موقت ژن Fol-SIX1 بروز یافت در حالیکه در ارقام Charentais Fom1(Ch-Fom1) و BG5384 (BG) تا 48 ساعت پس از تزریق نیز پاسخی بروز نیافت.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Functional analysis of Fol-SIX1 in resistant and susceptible Cucumis melo

نویسندگان [English]

  • Sahba Toosi 1
  • Farhad Shokouhifar 2
  • Saeed Malekzadeh Shafaroudi 3
  • Abdolreza Bagheri 4
1 M.Sc. student, Department of Biotechnology and Plant Breeding, Ferdowsi University of Mashhad, Mashhad, Iran
2 Assistant Professor, Research Center for Plant Sciences, Ferdowsi University of Mashhad, , Mashhad, Iran
3 Assistant Professor, Department of Plant Biotechnology and Breeding, Ferdowsi University of Mashhad, Mashhad, Iran
4 Professor, Department of Plant Biotechnology and Breeding, Ferdowsi University of Mashhad, Mashhad, Iran
چکیده [English]

Melon Vascular wilt caused by a soil-borne pathogen, Fusarium oxysporum f. sp melonis (Fom) is a distractive disease. The use of resistant cultivars is an effective way for controlling this fungus. Clear view about the interaction between virulence and resistant genes could provide applicable knowledge to design breeding programs. Fungus effector genes play a critical role during the plant-pathogen interactions. Exploring for the homologous of effector genes in different formae speciales started since some effector genes have been reported from Fusarium oxysporum f. sp. lycopersici (Fol). Recently, using bioinformatics assisted approach a homologous Fol-SIX1 effector gene has been reported from Fom. Here we performed a functional analysis study to reveal interactions between Fol-SIX1 and the melon differential varieties harboring defined R genes. Coding sequence of Fol-SIX1 from the pTS1 construct was subcloned in the pCAMBIA3301 binary vector in three steps and confirmed by colony PCR and digestion analysis. The accuracy of the expression construct, pCaS1 was evaluated by bidirectional sequencing using the PSh4-F2 and PSh51-R primers. Agroinjection-mediated transient expression approach was used to express Fol-SIX1 in leaves of the melon varieties. Functional analysis of Fol-SIX1 was performed by agroinjection of LBA4404 carrying pCaS1 into the leaves of melon lines. the leaves of Ch-T and Ch-Fom2 varieties developed a green dried symptom in response to transiently expressed Fol-SIX1 at 24 hours post injection (hpi), while Ch-Fom1 and BG lines did not responsive to the infection until 48 hpi.

کلیدواژه‌ها [English]

  • Agroinjection
  • Effector gene
  • Fol-SIX1
  • transient expression

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