Isolation Of Aluminum-tolerant Yeast And Identification Of Aluminum-responsive Genes In Cryptococcus Humicola

Aluminum in acid soils mainly on the biological toxic Al3+ form, therefore, aluminum toxicity is the main toxic factor effect the crop yield in acid soil. Study the aluminum-tolerance mechanism is basis to solve the aluminum toxicity problem. Microbial,growth fast, short period, is a better model to study the aluminum-tolerance mechanism. Separation the higher Al-tolerance of microorganisms, and analyzing the response genes under aluminum stress. So it can provide clues for Al-tolerance mechanism study, and also provide genetic resources for Al-sensitive crops.In this study, we isolate Al-tolerance strains, and identified Cryptococcus sp. response genes under aluminum stress by construct suppression subtractive hybridization library. The main results are as follows:Collection Baoshan Longling tea acidic soil to isolated aluminum tolerance rhizosphere microbes in pH3.5 and 20mmol.L-1 aluminum GM solid medium. Select 4 strong Al-tolerance strains 1-1,1-28,3-4 and 3-13 as a further study. Identification of fungi based on morphological and molecular evolution analysis of ITS and the D1/D2, successfully identified as Cryptococcus humicola (1-1), Cryptococcus rajasthanensis (1-28), Cryptococcus laurentii (3-4), Rhodotorula mucilaginosa (3-13). The ITS and D1/D2 sequences obtained gene sequences,and submitted to the GenBank database, gain access to Accession numbers, ITS are:1360946 (1-1),1361054 (1-28),1361190 (3-4),1361193 (3-13); D1/D2 were:1360505 (1-1),1361048(1-28),1361185 (3-4), 1361190(3-13).We analysis the Al-tolerance capacity of the four strains, the following results: the GM liquid medium in the 1-1 can grow in 200mmol.L-1,1-28,3-4,3-13 can be grown in 100mmol.L-1. In GM solid medium, four strains can be grown in 150mmol.L-1, which 1-1 and 1-28 still growth in 200mmol.L-1, but growth rate was significantly inhibited.Under 0,20,50,100,200mmol.L-1 condition, we count the apoptotic proportion of cryptococcus humicola by flow cytometry. With the increase of Al concentration, apoptosis cells increased; Apoptosis peak obviously appears at 100mmol.L-1; higher concentration of aluminum, the cells has become smaller. Determination content of residual aluminum in 20mmol.L-1 culture, the results show that the residual aluminum content over 98%. Absorption is not main Al-tolerance mechanisms in Cryptococcus humicola.Analysising Cryptococcus 1-1 physiological indicators under 20mmol.L-1 Aluminum stress. Data indicates that with the growth of the culture time, total protein content constant; PC content decreased; SOD activity decreased and subsequently up; soluble sugar content decreased and subsequently returned to normal; MDA rised.Construction of suppression subtractive hybridization cDNA library of Cryptococcus 1-1 under 20mmol.L-1 aluminum stress, library composed of selected 1,200 clones. PCR fragments detected mainly into between the 0.2～0.8 kb in the library. Selected from the SSH cDNA library clone insert fragments larger than 200bp were sequenced, sequencing analysis of 252 EST sequences obtained, excluding repeat sequence,141 different EST sequences was obtained. EST sequences of known function which accounts for 47%,6% of them associated with metabolism, energy metabolism associated with,16%, and defense-related stress,1%, associated with protein synthesis and processing,16%, signal transduction,2%, cytoskeleton-related, 6%; EST sequences of unknown function,19%; get 34% new EST sequence. Verified of library 14 genes by RT-PCR analysis, the results showed:ANTI, FAD, FBA, RGTP, GPD and VP these genes are upregulated under Al stress.