Sereening Of Resistance Pressure Of Porphyra Genetic Engineering And Preliminary Research On Porphyra Somaclonal Variation

Porphyra is one of the highest value of any cultivated seaweed, andhave already been one of the most important aquaculture species in China.And Porphyra is also the fundamental genetics research material for itssimple structure.It was often studied the antibiotic sensitivity of somatic cells ofporphyra gametophyte blades to develop Porphyra genetic engineering.However, the quantitative analysis was difficult because the somatic cellsof porphyra gametophyte blades were at different development stagesand pathways. This paper studied on the antibiotic sensitivity ofconchospore germLings in P. yezoensis by using three kinds of antibiotics,ampicillin, kanamycin and chloramphenicol. The results demonstrated that ampicillin could inhibit the growth and development of conchosporegermLings. It could slow down cell division and hinder the normaldevelopment of pigment body. After7days, the cell number of more than80%of conchospore germLings was less than15. After11days, themean length of conchospore germLings was shorter than that of controlgroup. And the mean length of conchospore germLings was shorter afterincreasing ampicillin concentration. The kanamycin could promote celldivision of conchospore germLings. After7days, the number of theconchospore germLings with over20cells was twice as many as that ofcontrol group. After11days, the mean length of conchospore germLingswas60%longer than that of control group. In addition, even if theconcentration was only100μg/mL, the promoting effect of kanamycinwas remarkable. After chloramphenicol treatment, more than90%ofconchospore germLings were dead when they only have2or3cells.When chloramphenicol concentration was higher than100μg/mL, theconchospore germLings were dead on the seventh day. When thechloramphenicol concentration was only50μg/mL, the conchosporegermLings were all dead on the eleventh day. It was concluded that thechloramphenicol was an effective selective pressure in porphyra geneticengineering.To expand genetic research methods in Porphyra haitanensis, we setup an appropriate AFLP analysis system by comparing different enzyme cut method. The results showed that EcoR I MseI or EcoRI/Taq I wasbetter for building AFLP fingerprinting of Porphyra haitanensis. Theproper enzyme cutting time is2-4h and the proper primer combination is1+2.We obtained some strains with excellent characters by somaclonalbreeding and found these characters were stable through the sexualreproduction. This paper detected the molecular level change ofsomaclonal variation by AFLP among the each generation of breedinggroup of thin thallus type in Porphyra haitanensis(HMC). Then we foundthe phenotype variation of HMC with the high level of DNApolymorphisms and the phenotype variation can be obtained by their F1generations. And we were also surprised to find character reversal in A4F1which exist in some high plant research. All these proved we can knowthe somaclonal variation in depth by AFLP.In addition, we obtain a second red variant from the red variant typein Porphyra haitanensis, and its red is brighter. The assay of the in vivoabsorption spectrum and the contents of main photosynthetic pigmentsshowed: the first and second absorption peak of the second variant raisedand the absorption peak of phycocyanin(PC) was lower. And that, thechlorophyll a(Chl. a) and phycobilioprotein(PE) contents of second redvariant were the same as the contents of red variant type, but thephycocyanin(PC) content of it decreased30%. So we assume the second variation may caused by the change of β-carotene and the decrease ofphycocyanin(PC). Meanwhile, we made a genetic analysis between thenatural red variant and its second red variant by AFLP. In48pairs ofprimer combinations, we obtained28pairs of primer combinations thatcould provide polymorphic bands. There were1627bands in all,including48polymorphic bands and the polymorphism proportion is5.16%. In addition, we also had a segment which was the homologousclip of bifunctional purine biosynthesis protein purH. Meanwhile，thespecific molecular characteristics of ZP-R and ZP-RR were obtained byZP8. These not only proved the reliability and stability of our AFLPsystem, also opened up the new way to study the formation mechanism ofcolor variation in Porphyra haitanensis.